Gene regulating aureobasidin sensitivity

ABSTRACT

The invention is directed to isolated DNAs having nucleic acid sequences which encode proteins which regulate aureobasidin sensitivity. Also disclosed are recombinant plasmids containing the DNAs, transformants containing the plasmids, and methods of producing the proteins.

This application is a continuation in part of application Ser. No.08/492,459 filed Jun. 29, 1995 now U.S. Pat. No. 6,015,689 which is acontinuation in part of application Ser. No. 08/243,403, filed May 16,1994, now abandoned.

FIELD OF THE INVENTION

This invention relates to a protein regulating a sensitivity to anantimycotic aureobasidin, a gene encoding this protein and to uses ofthe protein and gene.

DESCRIPTION OF RELATED ART

Systemic mycoses including candidiasis have increased with an increasein immunocompromised patients in recent years due to, for example, theextended use of immunosuppressive drugs and acquired immunodeficiencysyndrome (AIDS), and as opportunistic infection due to microbialsubstitution caused by the frequent use of widespectrum antibacterialantibiotics. Although drugs for treating mycoses such as amphotericin B,flucytosine and azole drugs (for example, fluconazole and miconazole)are now used to cope with this situation, none of them can achieve asatisfactory effect. Also, known diagnostic drugs are insufficient. Forcandidiasis, in particular, although there have been known severaldiagnostic drugs (for example, CAND-TEC for detection of candida antigenand LABOFIT for detection of D-arabinitol), none of them gives anysatisfactory results in specificity or sensitivity.

The reasons for the delay in the development of remedies and diagnosticdrugs for mycoses as described above are that fungi causing the mycosesare eukaryotic organisms similar to the host (i.e., man) and thus arenot largely different from man and that knowledges of fungi, inparticular, pathogenic fungi are insufficient. Therefore it is difficultto distinguish fungi from man or to selectively kill fungi, which isresponsible for the delay in the development of drugs for mycoses.

Recently, the application of genetic engineering techniques such asantisense or PCR to the treatment and diagnosis of mycoses has beenexpected. However known genes which are applicable thereto and/orproteins coded for by these genes are rare (PCT Pamphlet WO92/03455).Regarding pathogenic fungi, there have been cloned in recent years anacid protease gene, which has been assumed to participate in thepathogenicity of Candida albicans (hereinafter referred to simply as C.albicans) and Candida tropicalis (hereinafter referred to as C.tropicalis) causing candidiasis [B. Hube et al., J. Med. Vet. Mycol.,29, 129-132 (1991); Japanese Patent Laid-Open No. 49476/1993; and G.Togni et al., FEBS Letters, 286, 181-185 (1991)], a calmodulin gene ofC. albicans [S. M. Saporito et al., Gene, 106, 43-49 (1991)] and aglycolytic pathway enzyme enolase gene of C. albicans [P. Sundstrom etal., J. Bacteriology, 174, 6789-6799 (1991)]. However, each of thesegenes and proteins coded for thereby is either indistinguishable fromnonpathogenic fungi and eukaryotic organisms other than fungi or, ifdistinguishable therefrom, cannot serve as a definite action point forexhibiting any selective toxicity.

Aureobasidin [Japanese Patent Laid-Open No. 138296/1990, No. 22995/1991,No. 220199/1991, No. 279384/1993, and No. 65291/1994; J. Antibiotics, 44(9), 919-924, ibid., 44 (9), 925-933, ibid., 44 (11), 1187-1198 (1991)]is a cyclic depsipeptide obtained as a fermentation product of a strainAureobasidium pullulans No. R106. It is completely different instructure from other antimycotics. As Tables 1 and 2 show below,aureobasidin A, which is a typical aureobasidin compound, exerts apotent antimycotic activity on various yeasts of the genus Candidaincluding C. albicans which is a pathogenic fungus, Cryptococcusneoformans, Histoplasma capsulatum, Blastomyces dermatitidis and fungiof the genus Aspergillus and Penicillium (Japanese Patent Laid-Open No.138296/1990) but has an extremely low toxicity in mammal. Thus thiscompound is expected to be useful as an antimycotic being excellent inselective toxicity.

Hereinafter, Candida, Cryptococcus and Aspergillus will be abbreviatedrespectively as C, Cr. and A.

TABLE 1 Test Strain TIMM No. MIC(μg/ml) C. albicans 0136 ≦0.04 C.albicans var. stellatoidea 1308 ≦0.04 C. tropicalis 0312 0.08 C. kefyr0298 0.16 C. parapsilosis 0287 0.16 C. krusei 0270 ≦0.04 C.guilliermondii 0257 0.08 C. glabrata 1062 <0.04 Cr. neoformans 0354 0.63Cr. terreus 0424 0.31 Rhodotorula rubra 0923 0.63 A. fumigatus 0063 20A. clavatus 0056 0.16

TABLE 2 Test Strain TIMM No. MIC(μg/ml) A. nidulans 0112 0.16 A. terreus0120 5 Penicillium commune 1331 1.25 Trichophyton mentagrophytes 1189 10Epidermophyton floccosum 0431 2.5 Fonsecaea pedrosoi 0482 0.31 Exophialawerneckii 1334 1.25 Cladosporium bantianum 0343 0.63 Histoplasmacapsulatum 0713 0.16 Paracoccidioides brasiliensis 0880 0.31 Geotrichumcandidum 0694 0.63 Blastomyces dermatitidis 0126 0.31

Each of the existing antimycotics with a low toxicity shows only afungistatic action, which causes a clinical problem. In contrast,aureobasidin exerts a germicidal action. Although it has been requiredto clarify the mechanism of the selective toxicity of aureobasidin fromthese viewpoints, this mechanism still remains completely unknown.

As described in Canadian Patent Laid-Open No. 2124034, the presentinventors have previously found out that Saccharomyces cerevisiae(hereinafter referred to simply as S. cerevisiae) andSchizosaccharomyces pombe (hereinafter referred to simply as Schizo.pombe) are sensitive to aureobasidin. We have further mutated sensitivecells of S. cerevisiae or Schizo. pombe into resistant cells andsuccessfully isolated a gene capable of imparting a resistance toaureobasidin (a resistant gene) therefrom. We have furthermoresuccessfully isolated a gene capable of imparting aureobasidinsensitivity (a sensitive gene) from the corresponding sensitive cells.

We have also isolated a gene regulating aureobasidin sensitivity from C.albicans with the use of the gene regulating aureobasidin sensitivity ora part thereof as a probe. However no gene regulating aureobasidinsensitivity has been found in molds including those belonging to thegenus Aspergillus.

There have been known techniques for introducing useful genes intomonoploid fungal cells to be used in a laboratory, for example,Saccharomyces cerevisiae (hereinafter referred to simply as S.cerevisiae), Schizosaccharomyces pombe (hereinafter referred to simplyas Schizo. pombe) and Aspergillus nidulans (hereinafter referred tosimply as A. nidulans). Since the incorporation and fixation of plasmidDNAs into fungal cells are relatively scarcely successful, it isrequired to use selective markers in the identification oftransformants. In the most common case, selection can be achieved byintroducing an auxotrophic mutation into host cells. Examples of themutation generally employed in, for example, S. cerevisiae include ura3,leu2, trp1 and his3. A plasmid carries a wild type copy of one of thesegenes. Since the wild type copy on the plasmid is dominant over thechromosomal allele of the host, cells having the plasmid introducedthereinto can be screened in a minimal medium which contains no nutrientrequired by the auxotrophic host cells. Also there have been publishedsome reports, though in a small number, relating to the use of drugresistance in the screening of transformants. Namely, there have beenreported replication vectors and chromosome integration vectorscontaining genes which are resistant against antibiotics such as aneomycin homologue G418, hygromycin and cerulenin. A replication vectorhas a DNA replication origin acting in a cell. This plasmid is heldoutside the chromosome as a cyclic episome and continuously reduced at aratio of several percent with the proliferation of the cells. Anintegration vector is inserted into the chromosome of a host cell andthus held in a stable state. In this case, therefore, it is unnecessaryto further add a drug to the medium in order to exert the selectionfunction for maintaining the sequence of the vector.

In the case of industrial fungi, it is required to sustain the usefulcharacter, which has been imparted thereto, in a stable state. Achromosome integration vector is useful for this purpose.

Fungi have been widely applied to the production of liquors such assake, beer and wine and fermented foods such as miso (fermented soy beanpaste) and soy sauce. For breeding these fungi to be used for industrialpurposes, genetic engineering techniques are also highly effective inorder to impart useful characteristics thereto. Thus there have beenrequired selective markers which are usable in efficiently screeningtransformants. Industrial yeasts are usually di- or polyploid cells. Itis therefore difficult to introduce an auxotrophic marker, which iseffective in monoploid cells of, for example, yeasts to be used in alaboratory, into these industrial yeasts. In addition, since there is ahigh possibility that a mutagenesis induces mutation in other genes,accordingly, it is highly difficult to create a mutant having thedesired auxotrophic mutation alone introduced thereinto. The use of adrug resistance makes it possible to screen a stable transformant of anarbitrary yeast regardless of the number of chromosomes or theoccurrence of specific mutation. However many of these industrial fungiare insensitive to antibiotics such as G-418 and hygromycin, which makesit impossible to use genes resistant against these antibiotics therefor.Moreover, these resistant genes are genes or proteins derived frombacteria which are procaryotes, and none of them corresponding to thesegenes is present in fungi such as yeasts. The use of fungal cells havingthese foreign genes integrated therein is seriously restricted. Acerulenin resistant gene (PDR4) originating in S. cerevisiae is usablein the transformation of S. cerevisiae including brewing yeast. Howeverit also conferred resistances against drugs other than cerulenin, whichmight bring about some problems in the practical use. Therefore PDR4cannot fully satisfy the requirements for breeding industrial fungiincluding S. cerevisiae having improved characters in the future. Thusit has been required to develop drug resistant markers with the use ofgenes which are inherently carried by fungi.

There are a number of molds such as the ones of the genera Aspergillusand Penicillium. Some of these molds have been applied to foodmanufacturing (for example, brewing of liquors, soy sauce and miso,ripening of cheese, etc.) for a long time, while a number of them areimportant in the production of enzyme preparations or antibiotics.However, molds include not only these useful ones as described above butalso harmful ones such as those inducing plant diseases and thosecausing serious human diseases such as deep-seated mycosis. The recentdevelopment in genetic engineering techniques has made it possible notonly to breed useful strains but also to apply molds to novel purposes,for example, the production of a heterogenic protein. Also, analyses ofvital phenomena of molds are under way.

An object of the present invention is to find a gene, which encodes aprotein regulating aureobasidin sensitivity and which is useful ingenetic engineering techniques and in analyses of vital phenomena ofmolds from molds including those belonging to the genus Aspergillus andits functional derivative. That is to say, the present invention aims atrevealing a gene which encodes a protein regulating aureobasidinsensitivity or its functional derivative; providing a method for cloningthis gene and a protein regulating aureobasidin sensitivity encoded bythis gene or its functional derivative; providing the antisense DNA andthe antisense RNA of this gene; providing a nucleic acid probehybridizable with this gene and a method for detecting this gene byusing this nucleic acid probe; and providing a process for producing aprotein regulating aureobasidin sensitivity or its functional derivativeby using this gene.

Under these circumstances, the present invention further aims at findinga novel protein regulating aureobasidin sensitivity through theclarification of the mechanism of the selective toxicity to fungi ofaureobasidin. Accordingly, the present invention aims at finding a genecoding for a protein regulating aureobasidin sensitivity, providing aprocess for cloning this gene and the protein regulating aureobasidinsensitivity which is encoded by this gene, further providing anantisense DNA and an antisense RNA of this gene, providing a nucleicacid probe being hybridizable with this gene, providing a process fordetecting this gene with the use of the nucleic acid probe, providing aprocess for producing the protein regulating aureobasidin sensitivity byusing this gene and providing an antibody against the protein regulatingaureobasidin sensitivity, and a process for detecting the proteinregulating aureobasidin sensitivity by using this antibody.

In addition, the present invention aims at providing a novel chromosomeintegration vector capable of imparting a novel selective marker of adrug resistance to a fungal transformant, and a transformant transformedby this vector.

The present invention further aims at providing a protein capable ofimparting the aureobasidin resistance and acting as a selective markerwhich is usable in genetic engineering of fungi, and a DNA coding forthis protein.

SUMMARY OF THE INVENTION

The present invention may be summarized as follows. Namely, the firstinvention of the present invention relates to an isolated gene codingfor a protein regulating aureobasidin sensitivity, that is, a generegulating aureobasidin sensitivity. The second invention relates to aprocess for cloning a gene regulating aureobasidin sensitivity which ischaracterized by using the gene regulating aureobasidin sensitivity ofthe first invention or a part thereof as a probe. The third inventionrelates to a nucleic acid probe which is hybridizable with a generegulating aureobasidin sensitivity and comprises a sequence consistingof 15 or more bases. The fourth invention relates to an antisense DNA ofa gene regulating aureobasidin sensitivity. The fifth invention relatesto an antisense RNA of a gene regulating aureobasidin sensitivity. Thesixth invention relates to a recombinant plasmid having a generegulating aureobasidin sensitivity contained therein. The seventhinvention relates to a transformant having the above-mentioned plasmidintroduced thereinto. The eighth invention relates to a process forproducing a protein regulating aureobasidin sensitivity by using theabove-mentioned transformant. The ninth invention relates to an isolatedprotein regulating aureobasidin sensitivity. The tenth invention relatesto an antibody against a protein regulating aureobasidin sensitivity.The eleventh invention relates to a process for detecting a proteinregulating aureobasidin sensitivity by using the above-mentionedantibody. The twelfth invention relates to a process for detecting agene regulating aureobasidin sensitivity by the hybridization which ischaracterized by using the nucleic acid probe of the third invention ofthe present invention. The thirteenth invention relates to a process forscreening an antimycotic by using the above-mentioned transformant or aprotein regulating aureobasidin sensitivity. The fourteenth invention ofthe present invention relates to a chromosome integration vector for ahost fungus which is characterized by containing an aureobasidinresistant gene. This chromosome integration vector sometimes contains aforeign gene. The fifteenth invention relates to a process for producingan aureobasidin resistant transformant characterized by comprising:

(1) the step of obtaining a replication vector which contains anaureobasidin resistant gene,

(2) the step of cleaving the aureobasidin resistant gene in thereplication vector obtained in the above step at one site to give achromosome integration vector for a host fungus;

(3) the step of integrating the chromosome integration vector for a hostfungus obtained in the above step into the chromosome of the hostfungus; and

(4) the step of selecting a host which has been transformed into anaureobasidin resistant one in the presence of aureobasidin.

In this process for producing an aureobasidin resistant transformant,the replication vector sometimes contains a foreign gene. The sixteenthinvention relates to a transformant characterized by being one obtainedby the process of the fifteenth invention.

The seventeenth invention relates to a protein capable of impartingaureobasidin resistance, wherein at least the 240th amino acid residueAla in the protein capable of imparting aureobasidin sensitivityrepresented by SEQ ID No. 22 in the Sequence Listing has been replacedby another amino acid residue, or another protein capable of impartingaureobasidin resistance which has an amino acid sequence obtained bysubjecting the above-mentioned protein to at least one modificationselected from replacement, insertion and deletion of amino acidresidue(s) and shows a biological activity comparable to that of theabove-mentioned protein. The eighteenth invention relates to a DNA whichcodes for the protein capable of imparting the aureobasidin resistanceof the seventeenth invention.

The nineteenth invention relates to a gene originating in a mold whichencodes a protein regulating aureobasidin sensitivity or its functionalderivative. Namely, it relates to a gene regulating aureobasidinsensitivity obtained from a mold or a functional derivative thereof. Thetwentieth invention relates to a method for cloning a gene regulatingaureobasidin sensitivity and originating in a mold or its functionalderivative wherein the gene regulating aureobasidin sensitivity of thenineteenth invention or its functional derivative is employed as a probeeither as the whole or a part thereof. The twenty-first inventionrelates to a nucleic acid probe comprising a sequence consisting of atleast 15 bases which is hybridizable with a gene regulating aureobasidinsensitivity and originating in a mold or its functional derivative. Thetwenty-second invention relates to the antisense DNA of a generegulating aureobasidin sensitivity and originating in a mold or itsfunctional derivative. The twenty-third invention relates to theantisense RNA of a gene regulating aureobasidin sensitivity andoriginating in a mold or its functional derivative. The twenty-fourthinvention relates to a recombinant plasmid which contains a generegulating aureobasidin sensitivity and originating in a mold or itsfunctional derivative. The twenty-fifth invention relates to atransformant which has the plasmid of the twenty-fourth inventionintroduced thereinto. The twenty-sixth invention relates to a processfor producing a protein regulating aureobasidin sensitivity or itsfunctional derivative with the use of the above-mentioned transformant.The twenty-seventh invention relates to a protein regulatingaureobasidin sensitivity and originating in a mold or its functionalderivative. The twenty-eighth invention relates to a protein capable ofimparting the resistance to aureobasidin, wherein at least the aminoacid Gly at the position 275 of the protein imparting aureobasidinsensitivity represented by SEQ ID NO. 4 in the Sequence Listing has beenreplaced by another amino acid, or its functional derivative. Thetwenty-ninth invention relates to a DNA which encodes the protein of thetwenty-eighth invention capable of imparting the resistance toaureobasidin. The thirtieth invention relates to a method for detectinga gene regulating aureobasidin sensitivity by hybridization with the useof the nucleic acid probe of the twenty-first invention.

As described in Japanese Patent Application No. 106158/1994, the presentinventors have previously found out that fungi such as Schizo. pombe andS. cerevisiae and, further, mammalian cells such as mouse lymphoma EL-4cells, are sensitive to aureobasidin, as Table 3 shows.

TABLE 3 Test Strain or Cell MIC(μg/ml) Schizo. pombe 0.08 S. cerevisiae0.31 mouse lymphoma EL-4 10 mouse lymphoma L5178Y 100 NRK-49F 12.5

The present inventors have mutagenized a wild-type strain of Schizo.pombe or S. cerevisiae, sensitive to aureobasidin, to thereby giveresistant mutants. We have further successfully isolated a gene capableof confering aureobasidin resistance (a resistant gene) from theseresistant mutants and another gene capable of imparting aureobasidinsensitivity (a sensitive gene) from the corresponding sensitive cells.Furthermore, we have disclosed the existence of a protein encoded byeach of these genes. By culturing cells which have been transformed byintroducing the above-mentioned gene, we have succeeded in theexpression of this gene. Furthermore, we have successfully found out anovel gene regulating aureobasidin sensitivity from another fungus beingsensitive to aureobasidin by using a DNA fragment of the above-mentionedgene as a probe. In addition, we have clarified that the gene regulatingaureobasidin sensitivity is essentially required for the growth of thecells and found out that the detection of this gene or a protein whichis a gene product thereof with an antibody enables the diagnosis ofdiseases caused by these cells, for example, mycoses induced by fungi,and that an antisense DNA or an antisense RNA, which inhibits theexpression of the gene regulating aureobasidin sensitivity beingcharacteristic to the cells, is usable as a remedy for diseases causedby these cells, for example, mycoses induced by fungi, thus completingthe present invention. The present inventors have also succeeded in theexpression of this gene by preparing a replication vector containingthis gene and incubating cells transformed by using this vector. Byusing a DNA fragment of this gene as a probe, they have furthersuccessfully found a novel gene regulating the aureobasidin sensitivityfrom another fungus which is sensitive to aureobasidin.

The pathogenic fungi listed in Tables 1 and 2 and fungi and mammaliancells listed in Table 3, each having a sensitivity to aureobasidin; eachcarries a protein regulating aureobasidin sensitivity and a gene codingfor this protein. The term “a protein regulating aureobasidinsensitivity” as used herein means a protein which is contained in anorganism, particularly a fungus, having a sensitivity to aureobasidin.This protein is required for the expression of the sensitivity orresistance to aureobasidin. As a matter of course, a protein having 35%or more homology with the above-mentioned protein and having a similarfunction is also a member of the protein regulating aureobasidinsensitivity according to the present invention. Furthermore, proteinsobtained by modifying these proteins by the genetic engineeringprocedure are members of the protein regulating aureobasidin sensitivityaccording to the present invention. A gene regulating aureobasidinsensitivity means a gene which codes for such a protein regulatingaureobasidin sensitivity as those described above and involves both ofsensitive genes and resistant genes.

The first invention of the present invention relates to a generegulating aureobasidin sensitivity. This gene can be isolated in thefollowing manner. First, aureobasidin sensitive cells (a wild-typestrain) are mutagenized to thereby induce a resistant strain. Fromchromosome DNA or cDNA of this resistant strain, a DNA library isprepared and a gene capable of confering resistance (a resistant gene)is cloned from this library. Then a DNA library of a wild strain isprepared and a DNA molecule being hybridizable with the resistant geneis isolated from this library and cloned. Thus a sensitive gene can beisolated.

The mutagenesis is performed by, for example, treating with a chemicalsuch as ethylmethane sulfonate (EMS) orN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or by ultraviolet or otherradiation. The cell that has acquired the resistance can be screened byculturing the mutagenized cells in a nutritional medium containingaureobasidin at an appropriate concentration under appropriateconditions. The resistant strain thus obtained may vary depending on themethod and conditions selected for the mutagenesis. Also, strainsdiffering in the extent of resistance from each other can be separatedby changing the aureobasidin concentration or a temperature-sensitiveresistant strain can be isolated by changing the temperature in the stepof screening. There are a number of mechanisms of resistance toaureobasidin. Accordingly, a number of resistant genes can be isolatedby genetically classifying these various resistant strains. In the caseof a yeast, the classification may be performed by the complementationtest. Namely, resistant strains are prepared from haploid cells. Next,diploid cells can be obtained by crossing resistant strains differing inmating type from each other. Then spores formed from these diploids areexamined by the tetrad analysis.

As typical examples of the genes regulating aureobasidin sensitivity(named aur) according to the present invention, aur1 and aur2 genes maybe cited. Typical examples of the aur1 gene include spaur1 gene isolatedfrom Schizo. pombe and scaur1 gene isolated from S. cerevisiae, whiletypical examples of the aur2 gene include scaur2 gene isolated from S.cerevisiae. Now, resistant genes (spaur1^(R), scaur1^(R) and scaur2^(R))isolated from resistant mutants by the present inventors and sensitivegenes (spaur1^(S), scaur1^(S) and scaur2^(S)) isolated from sensitivewild-type strains will be described.

FIG. 1 shows a restriction enzyme map of the genes spaur1^(R) andspaur1^(S) regulating aureobasidin sensitivity, FIG. 2 shows arestriction enzyme map of scaur1^(R) and scaur1^(S) and FIG. 3 shows arestriction enzyme map of scaur2^(R) and scaur2^(S).

Schizo. pombe, which is sensitive to aureobasidin, is mutagenized withEMS and a genomic library of the resistant stain thus obtained isprepared. From this library, a DNA fragment containing a resistant gene(spaur1^(R)) and having the restriction enzyme map of FIG. 1 isisolated. This gene has a nucleotide sequence represented by SEQ ID No.15 in Sequence Listing. The amino acid sequence of a protein encoded bythis gene, which is estimated on the basis of this nucleotide sequence,is the one represented by SEQ ID No. 16 in Sequence Listing. By thehybridization with the use of this resistant gene as a probe, a DNAfragment containing a sensitive gene (spaur1^(S)) and having therestriction enzyme map of FIG. 1 is isolated from a sensitive strain.This gene has a nucleotide sequence represented by SEQ ID No. 17 inSequence Listing. The amino acid sequence of a protein encoded by thisgene, which is estimated on the basis of this nucleotide sequence, isthe one represented by SEQ ID No. 18 in Sequence Listing. A comparisonbetween the sequences of SEQ ID No. 17 and SEQ ID No. 15 reveals that amutation from G to T occurs at the base at the position 1053, while acomparison between the sequences of SEQ ID No. 18 and SEQ ID No. 16reveals that glycine at the residue 240 is converted into cysteine atthe amino acid level, thus giving rise to the resistance.

Also, S. cerevisiae, which is sensitive to aureobasidin, is mutagenizedwith EMS and genomic libraries of two resistant strains thus obtainedare prepared. From one of these libraries, a DNA fragment containing aresistant gene (scaur1^(R)) as a dominant mutant and having therestriction enzyme map of FIG. 2 is isolated, while a DNA fragmentcontaining a resistant gene (scaur2^(R)) and having the restrictionenzyme map of FIG. 3 is isolated from another library.

The nucleotide sequence of the coding region for the protein of thescaur1^(R) gene is the one represented by SEQ ID No. 19 in SequenceListing. The amino acid sequence of the protein encoded by this gene,which is estimated on the basis of the above nucleotide sequence, is theone represented by SEQ ID No. 20 in Sequence Listing. By thehybridization with the use of this resistant gene scaur1^(R) as a probe,a DNA fragment containing a sensitive gene (scaur1^(S)) and having therestriction enzyme map of FIG. 2 is isolated from a sensitive strain.This gene has a nucleotide sequence represented by SEQ ID No. 21 inSequence Listing. The amino acid sequence of a protein encoded by thisgene, which is estimated on the basis of this nucleotide sequence, isthe one represented by SEQ ID No. 22 in Sequence Listing. A comparisonbetween the sequences of SEQ ID No. 21 and SEQ ID No. 19 reveals that amutation from T to A occurs at the base at the position 852, while acomparison between the sequences of SEQ ID No. 22 and SEQ ID No. 20reveals that phenylalanine at the residue 158 is converted into tyrosineat the amino acid level, thus giving rise to the resistance. The spaur1gene has a 58% homology with the scaur1 gene at the amino acid level.Thus it is obvious that they are genes coding for proteins havingsimilar functions to each other. When genes and proteins beinghomologous in sequence with the spaur1 and scaur1 genes and with theproteins encoded thereby are searched from a data base, none having ahomology of 35% or above is detected. Accordingly, it is clear thatthese genes and the proteins encoded thereby are novel molecules whichhave never been known hitherto.

By the hybridization with the use of the DNA fragment of the resistantgene scaur2^(R) as a probe, a DNA fragment containing a sensitive gene(scaur2^(S)) and having the restriction enzyme map of FIG. 3 is isolatedfrom a sensitive strain.

The nucleotide sequence of this sensitive gene is the one represented bySEQ ID No. 23 in Sequence Listing and the amino acid sequence of theprotein encoded by this gene, which is estimated on the basis of thisnucleotide sequence, is the one represented by SEQ ID No. 24 in SequenceListing. As the result of the homology search with the scaur2^(S) geneand the protein encoded thereby, it has been found out that cysticfibrosis transmembrane conductance regulator (CFTR) of mammals alone hasa homology as low as 31%. Compared with this CFTR, however, the parthaving a high homology is limited to the region around the domain of thenucleotide binding. It is therefore obvious that the protein encoded bythe scaur2^(S) gene is a protein which is completely different from CFTRin function and has never been known hitherto.

In order to prove the importance of the aur1 gene in the growth ofcells, genes for disrupting the aur1 as shown in FIG. 4 and FIG. 5, inwhich genes coding for orotidine-5′-phosphate decarboxylase (ura4⁺ inthe case of Schizo. pombe, while URA3 in the case of S. cerevisiae) havebeen introduced midway in the aur1 gene, are prepared. When these aur1disrupted genes are introduced into Schizo. pombe and S. cerevisiaerespectively, the cells having the aur1 disrupted genes cannot grow atall. Thus it has been revealed that these genes and the proteins encodedthereby are essentially required for the growth of the yeast cells.

As the above examples clearly show, a gene regulating aureobasidinsensitivity can be isolated by using a organism having sensitivity toaureobasidin as a starting material and by carrying out the cloning withthe use of various mutagenesis methods and/or screening methodsdepending on the organisms or the methods. Also, genes beinghybridizable with the above-mentioned genes are involved in the scope ofthe first invention of the present invention. A gene regulatingaureobasidin sensitivity can be isolated by the following method. Thegenomic DNA library of an organism having sensitivity to aureobasidin isintegrated into, for example, a high-expression vector of a yeast andtransformed into the yeast. Then a clone having aureobasidin resistanceis selected from the transformants and DNA is recovered from this clone.Thus the resistant gene can be obtained. As a matter of course, genesobtained by modifying some part of the gene regulating aureobasidinsensitivity thus obtained by some chemical or physical methods areinvolved in the scope of the first invention of the present invention.

The second invention of the present invention relates to a process forcloning a gene regulating aureobasidin sensitivity which ischaracterized by using the gene regulating aureobasidin sensitivity ofthe first invention of the present invention or a part thereof as aprobe. Namely, by screening, by the hybridization method or thepolymerase chain reaction (PCR) method with the use of a part(consisting of at least 15 oligonucleotides) or the whole of the gene asobtained above, a gene coding for a protein having a similar functioncan be isolated.

For example, a pair of primers of SEQ ID No. 25 and SEQ ID No. 26 inSequence Listing are synthesized on the basis of the DNA nucleotidesequence of the spaur1^(R) gene represented by SEQ ID No. 15. Then PCRis performed by using cDNA of C. albicans, which is a pathogenic fungus,as a template with the use of the above-mentioned primers. The PCR iscarried out and the PCR products are electrophoresed on an agarose geland stained with ethidium bromide. In FIG. 6, the lanes 1, 2 and 3 showthe results obtained by using cDNA of C. albicans, cDNA of S. cerevisiaeand cDNA of Schizo. pombe as a template, respectively. As shown in FIG.6, a certain DNA fragment is specifically amplified.

By screening the genomic DNA library of C. albicans with the use of thisDNA fragment as a probe, a DNA molecule having a gene (caaur1), whichhas the same function as that of the spaur1 and scaur1 genes and havingthe restriction enzyme map of FIG. 7 is obtained. The nucleotidesequence of this caaur1 gene is the one represented by SEQ ID No. 27 inSequence Listing and the amino acid sequence of the protein encoded bythis gene, which has been estimated on the basis of the above nucleotidesequence, is the one represented by SEQ ID No. 28 in Sequence Listing.It has a high homology with the proteins encoded by the spaur1 andscaur1 genes.

By screening the genomic DNA library of C. albicans with the use of aDNA fragment comprising the whole length or a part of the scaur2^(S)gene represented by SEQ ID No. 23 in Sequence Listing as a probe, a DNAfragment containing gene (caaur2), which has the same function as thatof the scaur2 gene, and having the restriction enzyme map of FIG. 8 isobtained. The nucleotide sequence of a part of this caaur2 gene is theone represented by SEQ ID No. 29 in Sequence Listing and the amino acidsequence of the region encoded by this gene, which has been estimated onthe basis of this nucleotide sequence, is the one represented by SEQ IDNo. 30 in Sequence Listing. It has a high homology with thecorresponding region of the protein encoded by the scaur2 gene.

The third invention of the present invention relates to anoligonucleotide comprising 15 or more bases which serves as theabove-mentioned nucleic acid probe and is hybridizable with the generegulating aureobasidin sensitivity, for example, the DNA fragmenthaving the restriction enzyme map as shown in FIG. 1, FIG. 2 or FIG. 3.This nucleic acid probe is usable in, for example, the hybridization insitu, the identification of a tissue wherein the above-mentioned genecan be expressed, and the confirmation of the presence of a gene or mRNAin various vital tissues. This nucleic acid probe can be prepared byligating the above-mentioned gene or a gene fragment to an appropriatevector, introducing it into a bacterium, allowing it to replicate in thebacterium, extracting from a disrupted cell suspension, cleaving with arestriction enzyme capable of recognizing the vector-ligating site,electrophoresing and then excising from the gel. Alternatively, thisnucleic acid probe can be constructed by the chemical synthesis with theuse of a DNA synthesizer or gene amplification techniques by PCR on thebasis of the nucleotide sequence of SEQ ID Nos. 15, 17, 19, 21, 23, 27,29 or 35 in Sequence Listing. This nucleic acid probe can be labeledwith a radioisotope or a fluorescent substance to thereby elevate thedetection sensitivity during use.

The fourth invention of the present invention relates to an antisenseDNA of the above-mentioned gene regulating aureobasidin sensitivity,while the fifth invention of the present invention relates to anantisense RNA thereof. The introduction of the antisense DNA orantisense RNA into cells makes it possible to control the expression ofthe gene regulating aureobasidin sensitivity.

As examples of the antisense DNA to be introduced, antisense DNAscorresponding to the genes regulating aureobasidin sensitivity of SEQ IDNos. 15, 17, 19, 21, 23, 27, 29 or 35 in Sequence Listing and some partsthereof may be cited. SEQ ID No. 31 in Sequence Listing shows an exampleof this antisense DNA. It represents the sequence of an antisense DNA ofthe gene regulating aureobasidin sensitivity of SEQ ID No. 15 inSequence Listing. A fragment obtained by appropriately cleaving somepart of such an antisense DNA, and a DNA synthesized depending on suchan antisense DNA sequence may be used as the antisense DNA.

As examples of the antisense RNA to be introduced, antisense RNAscorresponding to the genes regulating aureobasidin sensitivity of SEQ IDNos. 15, 17, 19, 21, 23, 27, 29 or 35 in Sequence Listing and some partsthereof may be cited. SEQ ID No. 32 in Sequence Listing shows an exampleof this antisense RNA. It represents the sequence of an antisense RNA ofthe gene regulating aureobasidin sensitivity of SEQ ID No. 15 inSequence Listing. A fragment obtained by appropriately cleaving somepart of such an antisense RNA, an RNA synthesized depending on such anantisense RNA sequence, and an RNA prepared with RNA polymerase in an invitro transcription system by using the DNA corresponding to the generegulating aureobasidin sensitivity of SEQ ID No. 15 or SEQ ID No. 17 inSequence Listing or a part thereof may be used as the antisense RNA.

These antisense DNA and antisense RNA may be chemically modified so asto prevent degradation in vivo or to facilitate passage through a cellmembrane. A substance capable of inactivating mRNA, for example,ribozyme may be linked thereto. The antisense DNA and antisense RNA thusprepared are usable in the treatment of various diseases such as mycosesaccompanied by an increase in the amount of mRNA coding for a proteinregulating aureobasidin sensitivity.

The sixth invention of the present invention relates to a recombinantplasmid having a gene coding for a protein regulating aureobasidinsensitivity being integrated into an appropriate vector. For example, aplasmid, in which a gene regulating aureobasidin sensitivity gene hasbeen integrated into an appropriate yeast vector, is highly useful as aselection marker gene, since a transformant can be easily selectedthereby with the guidance of the chemical resistance by usingaureobasidin.

Also, the recombinant plasmid can be stably carried by, for example,Escherichia coli. Examples of vectors which are usable in this caseinclude pUC118, pWH5, pAU-PS, Traplex119 and pTV118. pAU-PS having thespaur1^(S) gene integrated therein is named pSPAR1. pWH5 having thespaur1^(S) gene integrated therein is named pSCAR1. pWHS having thescaur2^(R) gene integrated therein is named pSCAR². Traplex119 vectorhaving the caaur1 gene integrated therein is named pCAAR1. pTV118 vectorhaving a part of the caaur2 gene integrated therein is named pCAAR2N.Each of these recombinant plasmids is transformed into E. coli. It isalso possible to express these plasmids in an appropriate host. Such agene is reduced exclusively into the open reading frame (ORF) to betranslated into a protein by cleaving with an appropriate restrictionenzyme, if necessary, and then bound to an appropriate vector. Thus anexpression recombinant plasmid can be obtained. When E. coli is used asthe host, plasmids such as pTV118 may be used as a vector for theexpression plasmid. When a yeast is used as the host, plasmids such aspYES2 may be used as the vector. When mammalian cells are used as thehost, plasmids such as pMAMneo may be used as the vector.

The seventh invention of the present invention relates to a transformanthaving the above-mentioned recombinant plasmid which has been introducedinto an appropriate host. As the host, E. coli, yeasts and mammaliancells are usable. E. coli JM109 transformed by pSPAR1 having thespaur1^(S) gene integrated therein has been named and designated asEscherichia coli JM109/pSPAR1 and deposited at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology (1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, JAPAN), inaccordance with the Budapest Treaty under the accession number FERMBP-4485. E. coli HB101 transformed by pSCAR1 having the scaur1^(S) geneintegrated therein has been named and designated as Escherichia coliHB101/pSCAR1 and deposited at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology inaccordance with the Budapest Treaty under the accession number FERMBP-4483. E. coli HB101 transformed by pSCAR2 having the scaur2^(R) geneintegrated therein has been named and designated as Escherichia coliHB101/pSCAR2 and deposited at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology inaccordance with the Budapest Treaty under the accession number FERMBP-4484. E. coli HB101 transformed by pCAAR1 having the caaur1^(S) geneintegrated therein has been named and designated as Escherichia coliHB101/pCAAR1 and deposited at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology inaccordance with the Budapest Treaty under the accession number FERMBP-4482. E. coli HB101 transformed by pCAAR2N having a part of thecaaur2 gene integrated therein has been named and designated asEscherichia coli HB101/pCAAR2N and deposited at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology in accordance with the Budapest Treaty under the accessionnumber FERM BP-4481.

A transformant capable of expressing a protein regulating aureobasidinsensitivity can be obtained by transforming a expression recombinantplasmid into an appropriate host, as described above. For example, ayeast having a recombinant plasmid as shown in FIG. 9 introducedthereinto is usable for this purpose.

The eighth invention of the present invention relates to a process forproducing a protein regulating aureobasidin sensitivity which comprisesincubating a transformant according to the sixth invention of thepresent invention, which contains a gene coding for this protein, in anappropriate nutritional medium, allowing the expression of the protein,then recovering the protein from the cells or the medium and purifyingthe same. For the expression of the gene coding for this protein, E.coli, a yeast or mammalian cells are employed as a host. When the yeasthaving the recombinant plasmid of FIG. 9 is incubated in a mediumcontaining galactose, for example, the protein regulating aureobasidinsensitivity which is encoded by the scaur1^(S) gene can be expressed.

The ninth invention of the present invention relates to an isolatedprotein regulating aureobasidin sensitivity. As examples of such aprotein, those encoded by the above-mentioned spaur1, scaur1, scaur2,caaur1 and caaur2 genes can be cited.

The spaur1^(S) gene codes for a protein having an amino acid sequencerepresented by SEQ ID No. 18 in Sequence Listing, while the scaur1^(S)gene codes for a protein having an amino acid sequence represented bySEQ ID No. 22 in Sequence Listing. By the northern hybridization withthe use of a DNA fragment of the spaur1 gene as a probe, mRNAs aredetected from a sensitive strain (FIG. 10). Thus the expression of thespaur1 gene is confirmed.

FIG. 10 is an autoradiogram showing the results of the northernhybridization wherein mRNAs obtained from cells of a sensitive strain ofSchizo. pombe in the logarithmic growth phase (lane 1), cells of aresistant strain in the logarithmic growth phase (lane 2), cells of thesensitive strain in the stationary phase (lane 3) and cells of theresistant strain in the stationary phase (lane 4) are electrophoresed ona 1.2% agarose gel containing formaldehyde.

The tenth invention of the present invention relates to an antibodyagainst the above-mentioned protein regulating aureobasidin sensitivity.For example proteins having amino acid sequences of SEQ ID Nos. 16, 18,20, 22, 24, 28, 30 or 36 in Sequence Listing and peptides comprisingsome parts of these amino acid sequences may be used as an antigen. Theformer antigens can be prepared through the expression in a transformantfollowed by purification, while the latter antigens can be synthesizedon, for example, a marketed synthesizer. The antibody is produced by theconventional method. For example, an animal such as a rabbit isimmunized with the above-mentioned protein or a peptide fragmenttogether with an adjuvant to thereby give a polyclonal antibody. Amonoclonal antibody can be produced by fusing antibody-producing Bcells, which have been obtained by immunizing with an antigen, withmyeloma cells, screening hybridomas producing the target antibody, andincubating these cells. As will be described hereinafter, theseantibodies are usable in the treatment and diagnosis for animal andhuman diseases in which the above-mentioned proteins participate, suchas mycoses.

For example, a peptide corresponding to the part of the 103- to113-positions in the amino acid sequence of SEQ ID No. 22 is synthesizedon a synthesizer and then bound to a carrier protein. Then a rabbit isimmunized therewith and thus a polyclonal antibody is obtained. In thepresent invention, keyhole limpet hemocyanin (KLH) is used as thecarrier protein. Alternatively, bovine serum albumin and ovalbumin areusable therefor.

The eleventh invention of the present invention relates to a process fordetecting a protein regulating aureobasidin sensitivity by using theabove-mentioned antibody. The detection can be carried out by detectingthe binding of the antibody to the protein or measuring the amount ofbinding. For example, the protein or the cells producing the same can bedetected by treating with a fluorescence-labeled antibody and thenobserving under a fluorescence microscope. The amount of the antibodybound to the protein can be measured by various known methods. Forexample, S. cerevisiae cells are stained by the immunofluorescentantibody technique by using the above-mentioned antibody and a secondaryantibody such as FITC-labeled anti-rabbit antibody. Thus it is clarifiedthat the protein encoded by the scaur1 gene is distributed all over thecells. Further, a yeast having the recombinant plasmid of FIG. 9introduced thereinto is incubated in a medium containing glucose orgalactose. The cells thus obtained are disrupted with glass beads andproteins are solubilized. Then these proteins are separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the westernblotting is carried out in the conventional manner by using theabove-mentioned polyclonal antibody and peroxidase-labeled anti-rabbitantibody. Consequently, the protein encoded by the scaur1 gene can bedetected, as FIG. 11 shows.

FIG. 11 shows the results of the western blotting wherein the proteinsprepared from the cells obtained by the incubation in the presence ofglucose (lane 1) or galactose (lane 2) are subjected to SDS-PAGE. A mainband binding to the polyclonal antibody of the present invention isdetected at around 38 kDa.

The twelfth invention of the present invention relates to a process fordetecting a gene regulating aureobasidin sensitivity, for example, mRNAat the expression of a protein, by using the above-mentionedoligonucleotide as a nucleic acid probe. This process is applicable tothe diagnosis for various diseases, including mycoses, associated withan abnormal amount of mRNA coding for the protein. For example, nucleicacids are precipitated from disrupted cells and mRNA is hybridized witha radioisotope-labeled nucleic acid probe on a nitrocellulose membrane.The amount of binding can be measured by autoradiography (FIG. 10) orwith a scintillation counter.

The thirteenth invention of the present invention relates to a processfor efficient screening of a novel antimycotic by using the transformantof the seventh invention of the present invention or the proteinregulating aureobasidin sensitivity of the ninth invention of thepresent invention. For example, a drug exerting its effect on theprotein or the gene of the present invention can be efficiently foundout through a comparison of the activity on a transformant containing asensitive gene with the activity on a transformant containing aresistant gene or a comparison between the activities on transformantsdiffering in expression level from each other. Also, the screening canbe efficiently carried out by measuring the affinity for the protein ofthe present invention, for example, the activity of inhibiting thebinding of radiolabeled-aureobasidin to the protein.

As the above-mentioned examples clearly show, a gene regulating theaureobasidin sensitivity corresponding to each organism or each methodcan be isolated by employing a starting material, which is an organismhaving the sensitivity to aureobasidin, and effecting cloning byconducting various mutagenesis and/or screening treatments in the samemanner as the one described above. Moreover, genes hybridizable withthese genes can be isolated. As a matter of course, it is possible toprepare modified genes by partly altering the genes regulating theaureobasidin sensitivity obtained above by chemical, physical or geneticengineering techniques.

In the present invention, an aureobasidin resistant gene refers to agene which is capable of imparting the resistance to an antimycoticaureobasidin when integrated into a host fungus. This gene codes for aprotein imparting an aureobasidin resistance.

The aureobasidin resistant gene is exemplified typically by theabove-mentioned spaur1^(R) and scaur1^(R). Such a gene actspredominantly and the resistance conferred by this gene is selective toaureobasidin. That is to say, it does not cause any substantial changein the sensitivity to other drugs.

The aureobasidin resistant gene also involves genes which arehybridizable with spaur1^(R) and scaur1^(R) and impart the aureobasidinresistance to a host fungus (for example, genes prepared by partlyaltering the spaur1^(R) or scaur1^(R) gene by chemical, enzymatic,physical or genetic engineering techniques).

Furthermore, the aureobasidin resistant gene involves a gene coding fora protein, which has an amino acid sequence obtained by subjecting aprotein (Aur1^(R)p) capable of imparting the aureobasidin resistance toat least one modification selected from replacement, insertion anddeletion of amino acid residue(s) and shows the activity of impartingthe aureobasidin resistance.

The replacement, insertion and deletion of amino acid residue(s) fromAur1^(R)p can be effected by a site-specific mutagenesis. A DNA codingfor the isolated Aur1^(R)p or a DNA coding for the protein capable ofimparting the aureobasidin sensitivity (Aur1^(S)p) can be easilymodified by effecting at least one of the replacement, insertion anddeletion of nucleotide(s) and thus a novel DNA coding for a mutant ofAur1^(R)p can be obtained. Regarding the replacement, insertion anddeletion of amino acid residue(s), the conversion of the amino acid(s)is based on one which can be effected by genetic engineering techniqueswithout deteriorating the biological activity. In order to appropriatelyeffect the mutation on the residue at a specific site, the target codonis subjected to random mutagenesis and a mutant having the desiredactivity is screened from the ones thus expressed. The mutant obtainedby insertion involves a fused protein wherein Aur1^(R)p or its fragmentis bound to another protein or polypeptide at the amino terminal and/orthe carboxyl terminal of the Aur1^(R)p or its fragment via a peptidebond. In order to delete amino acid residue(s), it is also possible toreplace an arbitrary amino acid codon in the amino acid sequence with atermination codon by the gapped duplex method to thereby delete theregion on the carboxyl terminal side of the replaced amino acid residuefrom the amino acid sequence. Alternatively, a DNA coding for a protein,from which the amino terminal and/or carboxyl terminal regions in anarbitrary length have been deleted, can be obtained by the deletionmethod comprising degrading the coding DNA from the region(s)corresponding to the amino terminal and/or the carboxyl terminal of theamino acid sequence [Gene, 33, 103-119 (1985)] or a PCR method with theuse of primers containing an initiation codon and/or a terminationcodon. Known examples of the site-specific mutagenesis method includethe gapped duplex method with the use of oligonucleotide(s) [Methods inEnzymology, 154, 350-367 (1987)], the uracil DNA method with the use ofoligonucleotide(s) [Methods in Enzymology, 154, 367-382 (1987)], thenitrous acid mutation method [Proc. Natl. Acad. Sci. USA, 79, 7258-7262(1982)] and the cassette mutation method [Gene, 34, 315-323 (1985)].

The present inventors have found out that Aur1^(S)p represented by SEQID No. 22 in the Sequence Listing can be converted into Aur1^(R)p byreplacing the 240th residue Ala by another amino acid residue, thuscompleting the seventeenth and eighteenth inventions.

The Aur1^(R)p of the seventeenth invention is one wherein the 240thresidue Ala of Aur1^(S)p represented by SEQ ID No. 22 in the SequenceListing has been replaced by another amino acid residue. Other aminoacid residues may be replaced, inserted or deleted by using chemical,physical or genetic engineering techniques, so long as the biologicalactivity is not deteriorated thereby. The Aur1^(R)p of the seventeenthinvention may be appropriately prepared through genetic engineeringtechniques by using a DNA coding for Aur1^(S)p represented by SEQ ID No.47 in the Sequence Listing. Its biological activity can be assayed bymeasuring the activity of converting aureobasidin sensitive cells intoresistant cells. The Aur1^(R)p of the seventeenth invention is onehaving an enhanced activity of converting aureobasidin sensitive cellsinto resistant cells compared with Aur1^(R)p represented by SEQ ID No.20 in the Sequence Listing.

A preferable Aur1^(R)p is one having an enhanced activity of convertingaureobasidin sensitive cells into resistant cells compared withAur1^(R)p represented by SEQ ID No. 20 in the Sequence Listing. A DNAcoding for this Aur1^(R)p can be appropriately used in the presentinvention.

In an example of particularly preferable embodiment of Aur1^(R)p, amutant can be obtained by replacing the 240th residue Ala by Cys. Theamino acid sequence of an example of such a mutant is shown in SEQ IDNo. 42 in the Sequence Listing. This mutant is referred to as Aur1^(R)p(A240C). It is also possible to obtain a mutant wherein the 158thresidue Phe and the 240th residue Ala of Aur1^(S)p have been replacedrespectively by Tyr and Cys. The amino acid sequence of this mutant isshown in SEQ ID No. 43 in the Sequence Listing. This mutant is referredto as Aur1^(R)p (F158Y, A240C). Each of these mutants has a strongerability to impart aureobasidin resistance than that of the proteinrepresented by SEQ ID No. 40 in the Sequence Listing [Aur1^(R)p (F158Y)]wherein the 158th residue Phe of Aur1^(S)p has been replaced by Tyr.

The aureobasidin resistant gene to be used in the present invention isexemplified by the DNAs represented by SEQ ID Nos. 44 to 46 in theSequence Listing. The DNA represented by SEQ ID No. 46 in the SequenceListing is one coding for Aur1^(R)p (F158Y), the DNA represented by SEQID No. 44 in the Sequence Listing is one coding for Aur1^(R)p (A240C),and the DNA represented by SEQ ID No. 45 in the Sequence Listing is onecoding for Aur1^(R)p (F158Y, A240C).

A replication plasmid can be prepared by integrating a gene, which codedfor a protein regulating the aureobasidin sensitivity, into anappropriate vector. For example, a plasmid prepared by integrating anaureobasidin resistant gene into an appropriated yeast vector is highlyuseful as a selective marker gene, since a transformant can be easilyselected thereby depending on the drug resistance with the use ofaureobasidin. As the vector for yeasts, use can be made of ones ofYR_(p), YC_(p), YE_(p) and YI_(p) types.

Also, the replication plasmid can be stably carried by, for example,Escherichia coli, as described above. Examples of vectors which areusable in this case include pUC118, pWH5, pAU-PS, Traplex119 and pTV118.

The integration vector containing the aureobasidin resistant gene of thepresent invention is a linear vector which can be usually prepared bycleaving a replication plasmid containing the aureobasidin resistantgene into a linear form. The cleavage point in the replication plasmidwill be described hereinbelow.

FIG. 13 shows a process wherein an aureobasidin resistant gene in achromosome integration vector undergoes homologous recombination withthe host chromosome being homologous therewith (i.e., an aureobasidinsensitive gene) and thus aureobasidin sensitive cells are converted intoaureobasidin resistant cells. A replication plasmid containing theaureobasidin resistant gene is cleaved into a linear form at oneposition in the aureobasidin resistant gene sequence with an appropriaterestriction enzyme. The vector thus linearized undergoes homologousrecombination with the aureobasidin sensitive gene in the hostchromosome being homologous therewith. Thus the aureobasidin resistanceis imparted to the host cells. When the replication plasmid contains aforeign gene, then the aureobasidin resistance and the foreign gene areimparted to the host cells. For example, a replication vector pAUR1aarefor preparing a linear vector, which contains scaur1^(R) and humanacylamino acid releasing enzyme (AARE) described in Japanese PatentLaid-Open No. 254680/1991, is prepared. Escherichia coli JM109 strainhaving this vector introduced therein was named and indicated asEscherichia coli JM109/pAUR1aare and has been deposited at NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology under the accession number FERM P-14366. Tolinearize a replication vector, use can be effectively made of arestriction enzyme cleavage site which exists not in the target foreigngene moiety but in the aureobasidin resistant gene. In the cases of, forexample, scaur1^(R) originating in S. cerevisiae and spaur1^(R)originating in Schizo. pombe, restriction enzyme sites of StuI, etc. andBalI, etc. are usable respectively.

In a preferable form, the vector of the present invention may contain anaureobasidin resistant gene and a foreign gene and other genesoriginating in replication vectors may be eliminated therefrom.Promoters, terminators, etc. for expressing the aureobasidin resistantgene and the foreign gene may be selected depending on the characters ofthe host. As a matter of course, the promoter parts of the DNAsrepresented by SEQ ID Nos. 15 and 19 in the Sequence Listing can be usedas a promoter for expressing the function of the aureobasidin resistantgene. In the case of S. cerevisiae, use can be made of, for example,promoters of alcohol dehydrogenase gene (ADH1) andglyceraldehyde-3-phosphate dehydrogenase gene (GPD) and the terminatorof cytochrome C1 gene (CYC1). These promoters and terminators may bedifferent from those for expressing the aureobasidin resistant gene.

In the present invention, the term “foreign gene” refers to a gene whichis foreign to the host fungal cells, i.e., an alien gene. Examplesthereof include a nonfungal gene, a modified gene, a gene of a fungalspecies different from the host and a self-cloned gene. Moreparticularly, genes participating in fermentation, alcohol resistance,saccharification and the formation of taste components or aromacomponents fall within this category.

The fifteenth invention relates to a process for producing anaureobasidin resistant transformant. An aureobasidin resistanttransformant can be created by, for example, preparing a replicationvector containing the above-mentioned aureobasidin resistant gene,cleaving it at one position in the aureobasidin resistant gene in thereplication vector to give a linear chromosome integration vector for ahost fungus, adding this vector to aureobasidin sensitive host fungalcells under such conditions as to allow the transformation of the fungalcells, thus integrating the vector into the host chromosome, incubatingthe transformant in a medium suitable for the proliferation of the hostcells containing the antibiotic aureobasidin, and screening theaureobasidin resistant transformant thus proliferating. Thetransformation may be effected in accordance with publicly known methodssuch as the protoplast generation procedure, the lithium acetateprocedure or the electroporation procedure. The medium to be used hereinis not particularly restricted, so long as it is usable in theproliferation of fungi. Examples of such a medium commonly employedinclude Sabouraud's dextrose medium, a YPD medium, a czapek medium and aYNBG medium. The concentration of the aureobasidin added variesdepending on the host fungal cells having the sensitivity and usuallyranges from 0.05 to 80 μg/ml.

The transformant of the sixteenth invention can be obtained by theprocess of the fifteenth invention.

As an example of the transformant according to the present invention,Sake yeast Kyokai K-701 having scaur1^(R) and AARE gene integrated intothe chromosome has been deposited at National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology underthe accession number FERM P-1437. The transformant thus obtained byusing the chromosome integration vector of the present invention has anaureobasidin resistance imparted thereto and the foreign gene integratedthereinto which is held on the chromosome in a stable state. Thesecharacteristics make it highly useful in industrial uses, etc.

The Aur1^(R)p of the seventeenth invention can impart the aureobasidinresistance to monoploid yeasts and diploid yeasts, in particular,practically usable ones. Namely, it is highly useful in breeding S.cerevisiae which has been widely applied to liquors such as sake,shochu, beer and wine and fermented foods such as bread. Further, theAur1^(R)p of the seventeenth invention is applicable to fungi other thanS. cerevisiae and useful in, for example, breeding and geneticengineering application of other fungi.

For example, the Aur1^(R)p of the seventeenth invention is capable ofimparting the aureobasidin resistance to C. albicans. A vector havingthe DNA coding for this Aur1^(R)p is the first vector for geneticengineering uses provided for C. albicans.

It is known that C. albicans is a fungus causative of mycosis. With therecent increase in opportunistic infection, it has been needed toconduct studies for clarifying the causes of the pathogenicity. TheAur1^(R)p of the seventeenth invention and the above-mentioned vectorare highly useful in genetic studies on C. albicans.

The present inventors have further found out that molds such asAspergillus nidulans (hereinafter referred to simply as A. nidulans) andAspergillus fumigatus (hereinafter referred to simply as A. fumigatus)are sensitive to aureobasidin. Thus we have mutated sensitive cells ofA. nidulans into resistant cells and succeeded in the isolation of agene capable of imparting the resistance to aureobasidin (a resistantgene) from the corresponding resistant cells. Further, we have disclosedthe existence of a protein encoded by this gene. We have alsosuccessfully found novel genes regulating aureobasidin sensitivity fromaureobasidin sensitive A. nidulans and A. fumigatus by using a DNAfragment of the above-mentioned gene as a probe. Furthermore, we havefound out that the detection of this gene enables the diagnosis ofdiseases caused by these cells (for example, mycosis caused by fungi)and that the antisense DNA or antisense RNA, which inhibits theexpression of the gene regulating aureobasidin sensitivitycharacteristic of the cells, is usable as a remedy for diseases causedby these cells (for example, an antimycotic for mycosis).

The term “a protein regulating aureobasidin sensitivity” as used hereinmeans a protein which is contained in an organism, in particlar a mold,showing a sensitivity to aureobasidin. This protein is required forachieving a sensitivity or resistance to aureobasidin. The term “a generegulating aureobasidin sensitivity” means a gene which encodes such aprotein regulating aureobasidin sensitivity and a sensitive gene and aresistant gene fall within this category. The aureobasidin sensitivityof an organism varies depending on the molecular structure or amount ofsuch a protein or gene regulating aureobasidin sensitivity carried bythe organism.

The term “a functional derivative of the protein or gene regulatingaureobasidin sensitivity” as used herein means one which has abiological activity substantially comparable to that of the protein orDNA regulating aureobasidin sensitivity. It include fragments, variants,mutants, analogs, homologs and chemical derivatives. A variant means onewhich is substantially analogous to the whole protein or a fragmentoriginating therein in structure and/or function. That is to say, onemolecule which is essentially analogous to another in activity isregarded as a mutant, even though these two molecules are different inmolecular structure or amino acid sequence from each other. Thefunctional derivatives include proteins showing an amino acid sequencewith at least one modification selected from among replacement,insertion and deletion of amino acid residue(s) and having a comparablebiological activity and genes encoding these. The protein regulatingaureobasidin sensitivity may be subjected to the replacement, insertionand deletion of amino acid residues by a site-specific mutagenesis. Theisolated DNA encoding the protein regulating aureobasidin sensitivitycan be easily subjected to at least one modification selected from amongreplacement, insertion and deletion of nucleotides and thus a novel DNAencoding the protein regulating aureobasidin sensitivity and itsfunctional derivatives can be obtained.

Regarding the replacement, insertion and deletion of amino acidresidues, one or more amino acids can be converted by geneticengineering techniques and those suffering from no injury to thebiological activity should be selected. To properly effect a mutation onthe residue at a specified site, mutagenesis is performed at random onthe target codon and a mutant having the desired activity is screenedfrom the ones thus expressed. The mutant obtained by insertion involvesa fused protein wherein the protein regulating aureobasidin sensitivityor its functional derivative or a fragment thereof is bound via apeptide bond to another protein or polypeptide at the amino terminaland/or the carboxy terminal of the protein regulating aureobasidinsensitivity or its functional derivative or a fragment thereof. Todelete amino acid residue(s), an arbitrary amino acid codon in the aminoacid sequence may be replaced by a termination codon by thesite-specific mutagenesis. Thus the region on the carboxy terminal sideof the replaced amino acid residue can be deleted from the amino acidsequence. Alternatively a DNA coding for a protein, from which the aminoterminal and/or carboxy terminal regions in an arbitral length have beendeleted, can be obtained by the deletion method comprising degrading acoding DNA from the region(s) corresponding to the amino terminal and/orthe carboxy terminal of the amino acid sequence [Gene, 33, 103-119(1985)] or a PCR method with the use of primers containing an initiationcodon and/or a termination codon. Known examples of the site-specificmutagenesis method include the gapped duplex method with the use ofoligonucleotide(s) [Methods in Enzymology, 154, 350-367 (1987)], theuracil DNA method with the use of oligonucleotide(s) [Methods inEnzymology, 154, 367-382 (1987)], the nitrous acid mutation method[Proc. Natl. Acad. Sci. USA, 79, 7258-7262 (1982)] and the cassettemutation method [Gene, 34, 315-323 (1985)].

The nineteenth invention relates to a gene regulating aureobasidinsensitivity obtained from a mold exemplified by one belonging to thegenus Aspergillus or its functional derivative. In order to isolate thisgene, aureobasidin sensitive cells are first subjected to a mutagenesisto thereby derive a resistant strain therefrom. Then a DNA library isprepared from the chromosome DNAs or cDNAs of this resistant strain anda gene capable of imparting the resistance (a resistant gene) is clonedfrom this library. Similarly, a DNA library of a sensitive strain isprepared and DNA molecules hybridizable with the resistant gene areisolated and cloned. Thus a sensitive gene can be isolated.

The mutagenesis is performed by, for example, treating with a chemicalsuch as ethylmethane sulfonate (EMS) orN-methyl-N′-nitro-N-nitro-soguanidine (NTG) or by ultraviolet or otherradiation. A mutant that has acquired the resistance can be screened byculturing the mutagenized cells in a nutritional medium containingaureobasidin at an appropriate concentration under appropriateconditions. The resistant strain thus obtained may vary depending on themethod and conditions selected for the mutagenesis. It is furtherpossible to select strains differing in the extent of resistance byvarying the aureobasidin concentration at the screening. It is alsopossible to select a temperature-sensitive resistant strain by varyingthe temperature at the screening. Since there are two or more mechanismsof the resistance to aureobasidin, two or more resistant genes can beisolated by genetically classifying these resistant strains.

The genes regulating aureobasidin sensitivity of molds belonging to thegenus Aspergillus of the present invention include a gene anaur1^(R)isolated from a resistant mutant of A. nidulans, a gene anaur1^(S)isolated from a sensitive strain of A. nidulans and a gene afaur1^(S)isolated from a sensitive strain of A. fumigatus.

The attached FIG. 15 shows the restriction enzyme map of the genomic DNAof the gene anaur1^(R) regulating aureobasidin sensitivity andoriginating in a mold of Aspergillus, FIG. 16 shows the restrictionenzyme map of the cDNA of the gene anaur1^(S) and FIG. 17 shows therestriction enzyme map of the cDNA of the gene afaur1^(S).

A. nidulans sensitive to aureobasidin is mutagenized by UV irradiationand a genomic library of the resistant strain thus obtained is prepared.From this library, a DNA fragment containing a resistant gene(anaur1^(R)) and having the restriction enzyme map of FIG. 15 isisolated. This gene has a DNA sequence represented by SEQ ID NO. 1 inthe Sequence Listing. The amino acid sequence of a protein encoded bythis gene, which is estimated on the basis of this DNA sequence, is theone represented by SEQ ID NO. 2 in the Sequence Listing. By thehybridization with the use of this resistant gene, a cDNA fragmentcontaining a sensitive gene (anaur1^(S)) and having the restrictionenzyme map of FIG. 16 is isolated from a cDNA library of a sensitivestrain. This sensitive gene has a DNA sequence represented by SEQ ID NO.3 in the Sequence Listing. The amino acid sequence of a protein encodedby this gene, which is estimated on the basis of this base sequence, isthe one represented by SEQ ID NO. 4 in the Sequence Listing. Acomparison between the sequences of SEQ ID NO. 3 and SEQ ID NO. 1reveals that the genomic DNA has one intron (intervening sequence)ranging from the base at the position 1508 to the one at the position1563 in SEQ ID NO. 1. Further, G at the position 1965 in SEQ ID NO. 1has been mutated into T. A comparison between the sequences of SEQ IDNO. 4 and SEQ ID NO. 2 reveals that the amino acid glycine at theposition 275 has been mutated into valine at the amino acid level, thusgiving the resistance. The nineteenth invention also involves genesconstructed by chemically or physically altering a part of the genes ofthe present invention which regulate aureobasidin sensitivity andoriginate in molds.

The twentieth invention relates to a method for cloning a generegulating aureobasidin sensitivity and originating in a mold such asone of the genus Aspergillus or its functional derivative. This methodcomprises using the gene of the nineteenth invention regulatingaureobasidin sensitivity and originating in a mold, its functionalderivative, or a part of the same as a probe. That is to say, a geneencoding a protein having a comparable function can be isolated by thehybridization method or the polymerase chain reaction (PCR) method withthe use of the whole or a part of the gene (consisting of at least 15oligonucleotides) obtained above as a probe.

To examine a region appropriately usable as the above-mentioned probe,the present inventors have compared the amino acid sequence of theprotein encoded by the gene anaur1^(S) of the present invention (SEQ IDNO. 4 in the Sequence Listing) and the amino acid sequence of theprotein encoded by the gene afaur1^(S) of the present invention (SEQ IDNO. 5 in the Sequence Listing) with the amino acid sequence of theprotein encoded by an aureobasidin sensitive gene (scaur1^(S))originating in S. cerevisiae (SEQ ID NO. 6), the amino acid sequence ofthe protein encoded by another aureobasidin sensitive gene (spaur1^(S)originating in Schizo. pombe (SEQ ID NO. 7) and the amino acid sequenceof the protein encoded by a gene regulating aureobasidin sensitivity(caaur1) originating in C. albicans (SEQ ID NO. 8), each described inCanadian Patent No. 2124034. As a result, no homology is observed as thewhole. However, it has been revealed for the first time that there is acharacteristic sequence having been conserved in common in theseheterogenous genes regulating aureobasidin sensitivity. This conversedsequence has been very well conserved (homology: 80% or above) and iscomposed of at least eight amino acid residues, which corresponds to asufficiently long length to be used as a probe. FIG. 18 shows acomparison among the amino acid sequences represented by SEQ ID NOs. 4to 8 wherein three sequences (Box-1 to Box-3) named “Box sequences” bythe inventors correspond to the conserved sequence. Thus, a generegulating aureobasidin sensitivity and originating in mold or itsfunctional derivative can be cloned by using a primer or a probeconstructed from the amino acid sequence of Box 1, 2 or 3 respectivelyrepresented by SEQ ID NOs. 9, 10 or 11 in the Sequence Listing.

The amino acid sequences given in five rows in FIG. 18 correspondrespectively to SEQ ID NO. 4 (the top row), SEQ ID NO. 5 (the secondrow), SEQ ID NO. 6 (the third row), SEQ ID NO. 7 (the fourth row) andSEQ ID NO. 8 (the bottom row).

The target gene encoding the protein regulating aureobasidin sensitivityor its functional derivative may be obtained by hybridization in, forexample, the following manner. First, chromosomal DNAs obtained from thetarget gene source or cDNAs constructed from mRNAs with the use of areverse transcriptase are connected to a plasmid or a phage vector inaccordance with the conventional method and introduced into a host tothereby prepare a library. After incubating this library on a plate, thecolonies or plaques thus formed are transferred onto a nitrocellulose ornylon membrane and the DNAs are denatured and thus immobilized on themembrane. This membrane is incubated in a solution containing a probewhich has been preliminarily labeled with radio isotope ³²p, etc. (Theprobe to be used herein may be a gene encoding the amino acid sequencerepresented by SEQ ID NO. 4 in the Sequence Listing or a part of thesame. For example, use can be made of the gene represented by SEQ ID NO.3 in the Sequence Listing or a part of the same. It is appropriate touse therefor a base sequence which is composed of at least 15 bases andencodes one of the amino acid sequences represented by SEQ ID NOs. 9 to11 in the Sequence Listing or a part of the same.) Thus DNA hybrids areformed between the DNAs on the membrane and the probe. For example, themembrane having the DNAs immobilized thereon is hybridized with theprobe in a solution containing 6×SSC, 1% of sodium lauryl sulfate, 100μg/ml of salmon sperm DNA and 5×Denhardt's solution (containing bovineserum albumin, polyvinylpyrolidone and Ficoll each at a concentration of0.1%) at 65° C. for 20 hours. After the completion of the hybridization,nonspecifically adsorbed matters are washed away and clones forminghybrids with the probe are identified by autoradiography, etc. Into theclone thus obtained, a gene encoding the target protein has beenincluded.

It is confirmed whether or not the obtained gene is the one encoding thetarget protein regulating aureobasidin sensitivity or its functionalderivative, after the DNA sequence of the obtained gene is identifiedby, for example, the following method.

A clone obtained by the hybridization may be sequenced in the followingmanner. When the recombinant all is Escherichia coli, it is incubated ina test tube, etc. and the plasmid is extracted by a conventional method.Then it is cleaved with restriction enzymes and an insert thus excisedtherefrom is subcloned into an M13 phage vector, etc. Next, the basesequence is identified by the dideoxy method. When the recombinant is aphage, the base sequence can be identified fundamentally by the samesteps. These fundamental experimental procedures to be used from thecell culture to the DNA sequencing are described in, for example,Molecular Cloning, A Laboratory Manual, T. Maniatis et al., Cold SpringHarbor Laboratory Press (1982).

To confirm whether or not the obtained gene is the one encoding thetarget protein regulating aureobasidin sensitivity or its functionalderivative, the amino acid sequence thus identified is compared with theamino acid sequence represented by SEQ ID No. 4 in the Sequence Listingto thereby know the protein structure and amino acid sequence homology.

To examine whether or not the obtained gene sustains a sensitivity orresistance to aureobasidin, the obtained gene is transformed intosensitive cells and the aureobasidin sensitivity of the transformedcells thus obtained is determined to thereby reveal the activity of thegene. Alternatively, the activity can be determined by transforming theobtained gene into cells from which the activity has been eliminated bydisrupting or mutating the gene regulating aureobasidin sensitivity. Itis preferable that the above-mentioned gene to be transformed containssequences required for the expression (promoter, terminator, etc.) inthe upstream and/or downstream of the gene so as to enable theexpression in the cells transformed.

When the obtained gene fails to contain the whole region encoding theprotein regulating aureobasidin sensitivity or its functionalderivative, the base sequence of the whole region encoding the proteinregulating aureobasidin sensitivity or its functional derivative whichis hybridizable with the gene of the present invention encoding theprotein regulating aureobasidin sensitivity or its functional derivativecan be obtained by preparing synthetic DNA primers on the basis of thegene thus obtained, amplifying the missing region by PCR or furtherscreening a DNA library or a cDNA library with the use of a fragment ofthe obtained gene as a probe.

For example, a cDNA molecule having the restriction enzyme map of FIG.17, which contains a gene (afaur1^(s)) of A. fumigatus being comparablein function to the gene anaur1^(s), can be obtained by screening a cDNAlibrary of a pathogenic fungus A. fumigatus with the use of a DNAfragment of the PstI-EcoRI fragment (921 bp) of FIG. 16 as a probe. Thisgene has a base sequence represented by SEQ ID NO. 12 in the SequenceListing and the amino acid sequence of a protein encoded by this gene,which is estimated on the basis of this base sequence, is the onerepresented by SEQ ID NO. 5 in the Sequence Listing. When the genesanaur1^(s) and afaur1^(s) are compared, a homology of 87% is observed atthe amino acid level. Further, genomic DNAs prepared from Aspergillusniger (hereinafter referred to simply as A. niger) and Aspergillusoryzae (hereinafter referred to simply as A. oryzae) are subjected tothe Southern blotting analysis with the use of a DNA fragment of thegene anaur1^(s) as a probe. As a result, it is revealed that genesregulating aureobasidin sensitivity occur in A. niger and A. oryzae. Itis also possible to isolate genes regulating aureobasidin sensitivityfrom molds other than those belonging to the genus Aspergillus, forexample, ones of the genus Penicillium.

The twenty-first invention relates to the above-mentioned nucleic acidprobe, i.e., an oligonucleotide which is composed of at least 15 basesand hybridizable with a gene regulating aureobasidin sensitivity, forexample, a DNA fragment having a restriction enzyme map of FIGS. 15, 16or 17.

This nucleic acid probe is applicable to in situ hybridization, theconfirmation of a tissue wherein the above-mentioned gene is expressed,the confirmation of the existence of a gene or mRNA in various vitaltissues, etc. This nucleic acid probe can be prepared by ligating theabove-mentioned gene or its fragment to an appropriate vector,introducing it into a bacterium followed by replication, extracting withphenol, etc. from a disrupted cell solution, cleaving with restrictionenzymes capable of recognizing the ligation site with the vector,electrophoresing and excising from the electrophoresis gels.

Alternatively, this nucleic acid probe can be prepared by a chemicalsynthesis with the use of a DNA synthesizer or gene amplificationtechniques by PCR on the basis of each of the base sequences representedby SEQ ID NOs. 1, 3 and 12 in the Sequence Listing. Examples ofsequences appropriately usable as this nucleic acid probe include basesequences which are composed of at least 15 bases and encode the aminoacid sequences represented by SEQ ID NOs. 9 to 11 in the SequenceListing or a part of the same. To elevate the detection sensitivity, thenucleic acid probe may be labeled with a radioisotope or a fluorescentsubstance.

The twenty-second invention relates to the antisense DNA of theabove-mentioned gene regulating aureobasidin sensitivity and originatingin a mold, while the twenty-third invention relates to the antisense RNAthereof. By introducing this antisense DNA or antisense RNA into cells,the expression of the gene regulating aureobasidin sensitivity can becontrolled.

As the antisense DNA to be introduced, use can be made of, for example,the corresponding antisense DNAs of the genes regulating aureobasidinsensitivity represented by SEQ ID NOs. 1, 3 and 12 in the SequenceListing or a part of the same. SEQ ID NO. 13 in the Sequence Listingshows an example of such an antisense DNA which corresponds to thesequence of the antisense DNA of the gene regulating aureobasidinsensitivity represented by SEQ ID NO. 1 in the Sequence Listing. As theantisense DNA, it is also possible to use fragments obtained byappropriately cleaving these antisense DNAs or DNAs synthesized on thebasis of the sequences of these antisense DNAs.

As the antisense RNA to be introduced, use can be made of, for example,the corresponding antisense RNAs of the genes regulating aureobasidinsensitivity represented by SEQ ID NOs. 1, 3 and 12 in the SequenceListing or a part of the same. SEQ ID No. 14 in the Sequence Listingshows an example of such an antisense RNA which corresponds to thesequence of the antisense RNA of the gene regulating aureobasidinsensitivity represented by SEQ ID NO. 1 in the Sequence Listing. As theantisense RNA, it is also possible to use fragments obtained byappropriately cleaving these antisense RNAs or RNAs synthesized on thebasis of the sequences of these antisense RNAs. For example, use can bemade of an RNA prepared by using the corresponding antisense RNA of thegene regulating aureobasidin sensitivity represented by SEQ ID NO. 1 or3 in the Sequence Listing and treating it with RNA polymerases in an invitro transcription system.

The antisense DNA and antisense RNA can be chemically modified so as tomake them hardly degradable in vivo and enable them to pass through cellmembrane. A substance capable of inactivating mRNA such as a ribozymemay be bound thereto. The antisense DNA and antisense RNA thus preparedare usable in the treatment of various diseases such as mycosis inassociation with an increase in the content of the mRNA which encodesthe gene regulating aureobasidin sensitivity or its functionalderivative.

The twenty-fourth invention relates to a recombinant plasmid wherein thegene of the nineteenth invention, which encode a protein regulatingaureobasidin sensitivity or its functional derivative and originates ina mold, has been integrated into an appropriate vector. For example, aplasmid wherein an aureobasidin resistant gene has been integrated intoan appropriate yeast vector is highly useful as a selective marker gene,since it makes it easy to select a transformant showing the drugresistance against aureobasidin.

Also, a recombinant plasmid can be stably carried by Escherichia coli,etc. Examples of the vector usable therefor include pUC118, pWH5,pAU-PS, Traplex119 and pTB118.

It is also possible to transform a mold by ligating the gene of thenineteenth invention which encodes a protein regulating aureobasidinsensitivity or its functional derivative and originates in a mold to anappropriate vector. When a plasmid such as pDHG25 [Gene, 98, 61-67(1991)] is employed as the vector, the DNA introduced into the mold canbe maintained therein in the state of the plasmid. When a plasmid suchas pSa23 [Agricultural and Biological Chemistry, 51, 2549-2555 (1987)]is employed as a vector, the DNA can be stably maintained in the stateof having been integrated into the chromosome of the mold. It isfurthermore possible to give a recombinant plasmid for gene expressionby reducing the gene of the present invention into the open readingframe (ORF) alone by cleaving it with appropriate restriction enzymesand by ligating it to an appropriate vector. To construct the plasmidfor expression, use can be made of a plasmid such as pTV118, etc. (whenEscherichia coli is employed as the host), pYE2, etc. (when a yeast isemployed as the host), pMAMneo, etc. (when mammal cells are employed asa host) or pTAex3, etc. (when a mold is employed as the host) as thevector.

The twenty-fifth invention relates to a transformant obtained byintroducing the above-mentioned recombinant plasmid into an appropriatehost. As the host, use can be made of Escherichia coli, yeasts, moldsand mammal cells. Escherichia coli JM109 transformed by a plasmid pANAR1which had the gene anaur1^(s) integrated thereinto was named Escherichiacoli JM109/pANAR1 and has been deposited at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology under the accession number FERM BP-5180.

The twenty-sixth invention relates to a process for producing a proteinregulating aureobasidin sensitivity or its functional derivative. Thisprocess comprises incubating a transformant having the recombinantexpression plasmid of the twenty-fourth invention, which contains a geneencoding this protein or its functional derivative, in an appropriatenutritional medium, recovering and purifying the protein thus expressedfrom the cells or the medium. To express the gene encoding this protein,use is made of Escherichia coli, a yeast, a mold or mammal cells as thehost.

The twenty-seventh invention relates to a protein regulatingaureobasidin sensitivity or its functional derivative. Examples thereofinclude those encoded by the above-mentioned genes anaur1^(R),anaur1^(s) and afaur1^(s) and having amino acid sequences representedrespectively by SEQ ID NOs. 2, 4 and 5.

As a matter of course, these proteins may have at least one modificationselected from among replacement, insertion and deletion by chemical,physical or genetic engineering techniques. It is also possible toconstruct an antibody against a protein regulating aureobasidinsensitivity by using the proteins having the amino acid sequencesrepresented by SEQ ID NOs. 2, 4 and 5 or a peptide fragment of a regioncorresponding to a part of such an amino acid sequence as an antigen.

The twenty-eighth invention relates to a protein capable of impartingaureobasidin resistance wherein at least the amino acid Gly at theposition 275 in the gene imparting aureobasidin sensitivity representedby SEQ ID NO. 4 in the Sequence Listing has been replaced by anotheramino acid. This invention also involves functional derivatives of thesame obtained by introducing at least one modification selected fromamong replacement, insertion and deletion by chemical, physical orgenetic engineering techniques thereinto without any injury to thebiological activity thereof. The protein of the present inventioncapable of imparting aureobasidin resistance may be appropriatelyprepared genetic engineeringly by using DNAs encoding the proteinscapable of imparting aureobasidin resistance represented by SEQ ID NOs.3 and 12 in the Sequence Listing. Its biological activity can bedetermined by measuring the activity thereof of converting aureobasidinsensitive cells into aureobasidin resistant cells.

The twenty-ninth invention relates to a DNA encoding the protein of thetwenty-eighth invention capable of imparting aureobasidin resistance. Italso involves DNAs obtained by introducing at least one modificationselected from among replacement, insertion and deletion of nucleotide(s)into the above-mentioned DNA. Such a modification may be easily effectedby a site-specific mutagenesis. These modified DNAs are employed inorder to produce mutated proteins.

The thirtieth invention relates to a method for detecting a generegulating aureobasidin sensitivity by hybridization with the use of anucleic acid probe. Examples of the nucleic acid probe usable hereininclude oligonucleotides which are composed of at least 15 bases andhybridizable selectively with the DNAs represented by SEQ ID NOs. 1, 3and 12 in the Sequence Listing and fragments thereof. It is appropriateto use therefor base sequences which encode the amino acid sequencesrepresented by SEQ ID NOs. 9 to 11 in the Sequence Listing or a part ofthe same and consist of at least 15 bases. By using such a nucleic acidprobe, DNAs or RNAs extracted from the target organism are subjected toSouthern hybridization or Northern hybridization to thereby give thegene of the target organism regulating aureobasidin sensitivity. Thenucleic acid probe is also usable in the confirmation of a tissuewherein the above-mentioned gene can be expressed, or the confirmationof the existence of the gene or mRNA in various vital tissues by in situhybridization.

This nucleic acid probe can be prepared by ligating the above-mentionedgene or its fragment to an appropriate vector, introducing it into abacterium followed by replication, extracting with phenol, etc. from adisrupted cell solution, cleaving with restriction enzymes capable ofrecognizing the ligation site with the vector, electrophoresing andexcising from the gel. Alternatively, this nucleic acid probe can beprepared by a chemical synthesis with the use of a DNA synthesizer orgene amplification techniques by PCR on the basis of each of the basesequences represented by SEQ ID NOs. 1, 3 and 12 in the SequenceListing. To elevate the detection sensitivity in use, the nucleic acidprobe may be labeled with a radioisotope or a fluorescent substance.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction enzyme map of the genes spaur1^(R) andspaur1^(S) regulating aureobasidin sensitivity.

FIG. 2 is a restriction enzyme map of scaur1^(R) and scaur1^(S).

FIG. 3 is a restriction enzyme map of scaur2^(R) and scaur2^(S).

FIG. 4 shows the structure of a DNA for disrupting the Schizo. pombespaur1^(S) gene.

FIG. 5 shows the structure of a DNA for disrupting the S. cerevisiaescaur1^(S) gene.

FIG. 6 shows the results of the detection of the aur1 gene caaur1carried by C. albicans by the PCR method.

FIG. 7 is a restriction enzyme map of the caaur1 gene carried by C.albicans.

FIG. 8 is a restriction enzyme map of the caaur2 gene.

FIG. 9 shows the structure of a plasmid YEpSCARW3 for expressing thescaur1 gene.

FIG. 10 shows the results of the northern hybridization of the spaur1gene of Schizo. pombe.

FIG. 11 shows the results of the detection of the scaur1 protein byusing an antibody.

FIG. 12 is a restriction enzyme map of pAR25.

FIG. 13 shows a construction of the vector which is a example of thepresent invention, and integration of the vector into the chromosome.

FIG. 14 shows a pattern of the southern hybridization afterelectrophoresis of the genomic DNA digested by a restriction enzyme.

FIG. 15 is a diagram showing the restriction enzyme map of the genomicDNA of a gene anaur1^(R) regulating aureobasidin sensitivity.

FIG. 16 is a diagram showing the restriction enzyme map of the cDNA of agene anaur1^(s) regulating aureobasidin sensitivity.

FIG. 17 is a diagram showing the restriction enzyme map of the cDNA of agene afaur1^(s) regulating aureobasidin sensitivity.

FIG. 18 is a diagram showing a comparison among the amino acid sequencesof proteins encoded by genes regulating aureobasidin sensitivity.

FIG. 19 is a diagram showing a relation among the genomic DNA, cDNA andprotein of a gene anaur1 regulating aureobasidin sensitivity.

FIG. 20 is a diagram showing the results of Northern hybridization ofgenes regulating aureobasidin sensitivity of A. nidulans and A.fumigatus.

FIG. 21 is a diagram showing the results of Southern hybridization whichindicate the detection of genes regulating aureobasidin sensitivity ofA. niger and A. oryzae.

To further illustrate the present invention in greater detail, thefollowing Examples will be given. However it is to be understood thatthe present invention is not restricted thereto.

EXAMPLES Example 1

Cloning of a gene regulating aureobasidin sensitivity originating infission yeast Schizo. pombe

1-a) Separation of aureobasidin-resistant mutant of Schizo. pombe

About 1×10⁸ cells of a Schizo. pombe haploid cell strain JY745 (matingtype h⁻, genotype ade6-M210, leu1, ura4-D18) exhibiting a sensitivity toaureobasidin at a concentration of 0.08 μg/ml were suspended in 1 ml ofa phosphate buffer containing 0.9% NaCl. Then the cells were mutagenizedwith EMS at a final concentration of 3% at 30° C. for 90 minutes. Afterneutralizing by adding 8 ml of 5% sodium thiosulfate, the cells thustreated were harvested by centrifugation (2500 r.p.m., 5 minutes),washed twice with 6 ml of physiological saline and then suspended in 2ml of a YEL medium (3% of glucose, 0.5% of yeast extract). Thesuspension was incubated at 30° C. for 5 hours under stirring and thenspreaded on a YEA plate (the YEL medium containing 1.5% of agar)containing 5 μg/ml of aureobasidin A. After incubating at 30° C. for 3to 4 days, two or three aureobasidin-resistant colonies were formed per1×10⁸ cells. After carrying out the mutagenesis several times, fiveclone mutants, i.e., THR01, THR04, THR05, THR06 and THR07 were obtained.These mutants were resistant to more than 25 μg/ml of aureobasidin A butthe same as the parent strain in the sensitivity to cycloheximide andamphotericin B. Therefore it is estimated that they are not mutantshaving a multiple drug resistance but ones having a resistance specificto aureobasidin.

1-b) Genetic analysis

Each of the above-mentioned resistant strains THR01, THR04, THR05, THR06and THR07 was crossed with normal cells of Schizo. pombe LH121 strain(mating type h⁺, genotype ade6-M216, ura4-D18) differing in mating type.Diploid cells obtained were examined about the resistance toaureobasidin. Similar to the resistant strains, the five diploids formedby crossing the resistant strains with the normal one were resistant to25 μg/ml of aureobasidin A, thus proving that these resistant mutationswere dominant. To perform the tetrad analysis, the above-mentioneddiploids were subsequently inoculated on an MEA medium (3% of maltextract, 2.5% of agar) for sporulation and incubated at 25° C. for 2days. Prior to the meiosis, the diploid cells replicated DNA on the MEAmedium and then underwent the meiosis to form asci each containing fourascospores of the haploid. These spores were separated with amicromanipulator and allowed to germinate on the YEA plate, followed bythe formation of colonies. Then the resistance to aureobasidin of thesecolonies was examined. Among four spores contained in an ascus, theseparation of the sensitivity versus the resistance showed 2:2. Thisresult indicates that the aureobasidin resistant mutation was induced bya mutation in single gene. Further, the complementation test wasperformed in order to confirm whether the resistant genes of theabove-mentioned five mutants were identical with each other or not. Forexample, a mutant of the mating type h⁺, which had been obtained bycrossing the mutant THR01 with the LH121 strain in the above tetradanalysis, was crossed with another variant THR04 (mating type h⁻) on theMEA plate as described above and, after sporulation, the tetrad analysiswas carried out. As a result, all of the colonies formed from fourascospores showed resistance to aureobasidin, which indicates that themutational genes of THR01 and THR04 are the same with each other.Similarly, the five mutants were examined and it was thus found out thatall mutations occurred on the same gene. This gene regulatingaureobasidin sensitivity is named spaur1, the normal gene (sensitivegene) is named spaur1^(S) and the mutational gene (resistant gene) isnamed spaur1^(R).

1-c) Preparation of genomic library of aureobasidin resistant strain

Genomic DNA was extracted and purified from the aureobasidin resistantstrain THR01 by the method of P. Philippsen et al. [Methods inEnzymology, 194, 169-175 (1991)]. The purified genomic DNA (8 μg) waspartially digested by treating with 5 U of a restriction enzyme HindIIIat 37° C. for 10 minutes, deproteinized with phenol/chloroform andprecipitated with ethanol. The partially digested DNA waselectrophoresed on a 0.8% agarose gel and DNA in the region of 3 to 15kb was extracted. The DNA thus obtained was ligated with a yeast-E. colishuttle vector pAU-PS (2 μg) which had been completely digested withHindIII by using a DNA ligation kit (manufactured by Takara Shuzo Co.,Ltd.) and then transformed into E. coli HB101. Thus a genomic library ofthe aureobasidin resistant strain was formed. E. coli containing thisgenomic library was incubated in 50 ml of an LB medium (1% of bactotrypton, 0.5% of bacto yeast extract, 0.5% of sodium chloride)containing 100 μg/ml of ampicillin and 25 μg/ml of tetracycline at 37°C. overnight. Then a plasmid was recovered and purified from the E. colicells.

1-d) Expression and cloning of aureobasidin resistant gene spaur1^(R)

The plasmid originating in the genomic library of the aureobasidinresistant strain as prepared above was transformed into a strain Schizo.pombe JY745 by the method of Okazaki et al. [Nucleic Acid Research, 18,6485-6489 (1990)]. The transformed cells were spreaded on a minimummedium SD plate [0.67% of yeast nitrogen base without amino acids(manufactured by Difco), 2% of glucose, 2% of agar] containing 75 μg/mlof adenine sulfate and 50 μg/ml of leucine. After incubating at 30° C.for 3 to 4 days, the colonies thus formed were replicated onto an SDplate containing 5 μg/ml of aureobasidin A, 75 μg/ml of adenine sulfateand 50 g/ml of leucine. It is conceivable that a colony propagated onthis plate may have the plasmid containing the aureobasidin resistantgene. This colony was inoculated into 5 ml of a liquid SD mediumcontaining 75 μg/ml of adenine sulfate and 50 μg/ml of leucine. Afterincubating at 30° C. for 2 days, the plasmid was recovered from thepropagated cells by the method of I. Hagan et al. [J. Cell Sci., 91,587-595 (1988)]. Namely, the cells were harvested from the culture (5ml) by centrifugation and then suspended in 1.5 ml of 50 mMcitrate/phosphate buffer containing 1.2 M of sorbitol and 2 mg/ml ofZymolyase. Then the suspension was maintained at 37° C. for 60 minutes.The cells were collected by centrifuging at 3,000 r.p.m. for 30 secondsand suspended in 300 μl of a TE [10 mM of Tris-HCl, pH 8, 1 mM of EDTA]solution. After adding 35 μl of 10% SDS, the mixture was maintained at65° C. for 5 minutes. After adding 100 μl of 5 M potassium acetate, themixture was allowed to stand in ice for 30 minutes. Then it wascentrifuged at 10,000 r.p.m. at 4° C. for 10 minutes and a plasmid DNAwas purified from the supernatant by using EASYTRAP™ (manufactured byTakara Shuzo Co., Ltd.).

This plasmid was transformed into E. coli HB101 and a plasmid DNA wasprepared from E. coli colonies formed on an LB medium containingampicillin and tetracycline. This plasmid, which contained a DNA of 4.5kb, was named pAR25. FIG. 12 shows the restriction enzyme map of the DNAof 4.5 kb in pAR25. To specify the gene region, HindIII fragments orSacI fragments of various sizes were subcloned into the pAU-PS vector.These DNAs were transformed into normal JY745 cells by theabove-mentioned method of Okazaki et al. and the acquisition ofaureobasidin resistance was examined. As a result, it is revealed that aHindIII-SacI 2.4 kb DNA fragment contains the spaur1^(R) gene. Therestriction enzyme map of this DNA segment containing the aureobasidinresistant gene spaur1^(R) is shown in FIG. 1. This fragment was clonedinto a pUC118 vector (named pUARS2^(R)) and then the DNA nucleotidesequence was identified (SEQ ID No. 1 in Sequence Listing). From thisnucleotide sequence, it is revealed that the spaur1^(R) gene code for aprotein having an amino acid sequence represented by SEQ ID No. 16 inSequence Listing.

1-e) Cloning of aureobasidin sensitive gene spaur1^(S)

By the same method as the one employed in the above c), genomic DNA wasextracted and purified from normal cells. After partially digesting withHindIII, a genomic library of the normal cells was constructed. An E.coli stock containing this library DNA was spreaded on an LB agar mediumcontaining ampicillin and tetracycline and incubated overnight at 37° C.The colonies thus formed were transferred onto a nylon membrane(Hybond™-N, manufactured by Amersham) and the colony hybridization wasperformed.

As a probe, the above-mentioned DNA fragment (2.4 kb) obtained bycleaving the spaur1^(R) gene with HindIII-SacI and labeled with [α-²³P]dCTP by using a random primer DNA labeling kit (manufactured by TakaraShuzo Co., Ltd.) was used. As the results of screening of 5×10⁴colonies, five clones being hybridizable with the probe were obtained.Plasmids were purified from E. coli cells of these five clones. As theresult of the cleavage with restriction enzymes, it was found out thatall of these clones contained the same DNA fragment of 4.5 kb (namedpARN1). The restriction enzyme map of the DNA of 4.5 kb in pARN1 wasidentical with that of pAR25 shown in FIG. 10. Therefore, a HindIII-SacI2.4 kb DNA fragment which was a region containing the spaur1^(S) genewas prepared from PARN1. Then it was cloned into the pAU-PS vector andthis plasmid was named pSPAR1.

By using this plasmid pSPAR1, a strain E. coli JM109 was transformed andthe transformant thus obtained was named and designated as Escherichiacoli JM109/pSPAR1. It has been deposited at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology in accordance with the Budapest Treaty under the accessionnumber FERM BP-4485. This DNA fragment containing the aureobasidinsensitive gene spaur1^(S) had the restriction enzyme map shown in FIG. 1and the DNA nucleotide sequence thereof was the one represented by SEQID No. 17 in Sequence Listing. Based on this nucleotide sequence, it hasbeen revealed that the spaur1^(S) gene codes for a protein having theamino acid sequence represented by SEQ ID No. 28 in Sequence Listingand, when compared with the resistant gene spaur1^(R), the amino acid atthe residue 240 has been changed from glycine into cysteine.

Example 2

Cloning of aureobasidin sensitive genes scaur1 and scaur2 originating inbudding yeast S. cerevisiae

2-a) Separation of aureobasidin resistant mutant of S. cerevisiae

A strain S. cerevisiae DKD5D (mating type a, genotype leu2-3 112, trp1,his3) having a sensitivity to aureobasidin at a concentration of 0.31μg/ml was mutagenized with EMS in the same manner as the one employed inthe case of Schizo. pombe. Then resistant mutants were separated on anagar plate of a complete nutritional medium YPD (1% of yeast extract, 2%of polypeptone, 2% of glucose) containing 5 μg/ml or 1.5 μg/ml ofaureobasidin A. After repeating the mutagenesis several times, 34 mutantclones were obtained. These mutants were resistant to more than 25 μg/mlof aureobasidin A and estimated as having not a multiple drug resistancemutation but a aureobasidin-specific resistance mutation.

2-b) Genetic analysis

Similar to the above-mentioned case of Schizo. pombe, the geneticanalysis using the tetrad analysis and the complementation test wasperformed. As a result, the genes could be classified into two types.These genes regulating aureobasidin sensitivity were named scaur1 andscaur2, the resistant genes isolated from the resistant mutant werenamed scaur1^(R) and scaur2R, and the sensitive genes isolated from thesensitive wild-type strain were named scaur1^(S) and scaur2^(S),respectively.

The R94A strain had a gene with dominant mutation (scaur1^(R)). It hasbeen further clarified that the scaur1 gene is located in theneighborhood of the met14 gene of the eleventh chromosome.

2-c) Preparation of genomic library of aureobasidin resistant strainhaving aureobasidin resistant gene scaur1^(R)

Genomic DNA was extracted and purified from the aureobasidin resistantstrain R94A by the above-mentioned method of P. Philippsen et al. Thepurified genomic DNA (8 μg) was partially digested by treating with 5 Uof a restriction enzyme HindIII at 37° C. for 10 minutes, deproteinizedwith phenol/chloroform and precipitated with ethanol. The partiallydigested DNA thus obtained was electrophoresed on a 0.8% agarose gel andDNA in the region of 3 to 15 kb was extracted. The DNA thus obtained wasligated with a yeast-E. coli shuttle vector pWH5 (2 μg) which had beencompletely digested with HindIII by using a DNA ligation kit and thentransformed into E. coli HB101. Thus a genomic library was formed. E.coli containing this genomic library was cultured in 50 ml of an LBmedium containing ampicillin and tetracycline at 37° C. overnight. Thena plasmid was recovered and purified from the E. coli cells.

2-d) Expression and cloning of aureobasidin resistant gene scaur1^(R)

The above-mentioned genomic library of the R94A strain was transformedinto S. cerevisiae SH3328 (mating type α, genotype ura3-52, his4, thr4,leu2-3•112) in accordance with the method of R. H. Schiestl et al.[Current Genetics, 16, 339-346 (1989)]. The transformed cells werespread on a minimum medium SD plate [0.67% of yeast nitrogen basewithout amino acids, 2% of glucose, 2% of agar] containing 25 μg/ml ofuracil, 35 μg/ml of histidine and 500 μg/ml of threonine. Afterincubating at 30° C. for 3 to 4 days, the colonies thus formed werereplicated onto a YPD agar plate containing 1.5 μg/ml of aureobasidin A.A colony thus formed was inoculated into 5 ml of a liquid YPD medium.After incubating at 30° C. for 2 days, a plasmid DNA was recovered fromthe propagated cells by the above-mentioned method of 1. Hagan et al.This plasmid was transformed into a yeast again and it was confirmedthat the obtained transformant had acquired aureobasidin resistance.This plasmid, which contained a DNA of 3.5 kb, was named pWTCR3. Neitherthe DNA fragment of 2.0 kb nor the DNA fragment of 1.5 kb obtained bycleaving with HindIII exhibited any aureobasidin resistant activityalone. Thus it is confirmed that the gene is contained in the DNAfragment of 3.5 kb. FIG. 2 shows the restriction enzyme map of this DNAfragment of 3.5 kb containing the aureobasidin resistant genescaur1^(R). The HindIII fragments of 1.5 kb and 2 kb were each clonedinto pUC118, followed by the determination of the DNA nucleotidesequence (SEQ ID No. 19 in Sequence Listing). From this nucleotidesequence, it has been revealed that the scaur1^(R) gene codes for aprotein having an amino acid sequence represented by SEQ ID No. 20 inSequence Listing.

2-e) Cloning of aureobasidin sensitive gene scaur1^(S) corresponding toaureobasidin resistant gene scaur1^(R)

By the same method as the one employed in the above Example 2-c),genomic DNA was extracted and purified from the parent strain S.cerevisiae DKD5D. After partially digesting with HindIII, the DNA wasligated with pWH5 and transformed into E. coli HB101. Thus a genomiclibrary of the normal cells was formed. An E. coli stock containing thislibrary DNA was spreaded on an LB agar medium containing ampicillin andtetracycline and incubated overnight at 37° C. The colonies thus formedwere transferred onto a nylon membrane (Hybond™-N) and the colonyhybridization was carried out. As a probe, the DNA fragment of 3.5 kbobtained in the above Example 2-d) and labeled with [α-³²P] dCTP byusing a random primer DNA labeling kit (manufactured by Takara ShuzoCo., Ltd.) was used. As the results of screening of 2×10⁴ colonies,seven clones being hybridizable with the probe were obtained. Plasmidswere purified from E. coli cells of these clones. As the result of thecleavage with restriction enzymes, one of these clones contained a DNAfragment of 3.5 kb. This DNA fragment had the restriction enzyme map ofFIG. 2 and thus judged as containing the scaur1^(S) gene. The plasmidcontaining this DNA fragment was named pSCAR1, while E. coli HB101having this plasmid introduced therein was named and designated asEscherichia coli HB101/pSCAR1. This strain has been deposited atNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology in accordance with the Budapest Treatyunder the accession number FERM BP-4483. The DNA fragment of 3.5 kbobtained by partially digesting pSCAR1 with HindIII was subcloned intopUC118 and the nucleotide sequence thereof was determined (SEQ ID No. 21in Sequence Listing). A comparison with the resistant gene indicatesthat the base at the position 852 has been changed from T into A and,due to this replacement, the amino acid has been converted fromphenylalanine into tyrosine (SEQ ID No. 22 in Sequence Listing).

2-f) Preparation of genomic library of aureobasidin resistant strainhaving aureobasidin resistant gene scaur2R

A genomic library was prepared from an aureobasidin resistant strainL22-8B by the same method as the one described in Example 2-c). E. colicontaining this genomic library was cultured in an LB medium (50 ml)containing ampicillin and tetracycline at 37° C. overnight. Thenplasmids were recovered and purified from the E. coli cells.

2-g) Expression and cloning of aureobasidin resistant gene scaur2^(R)

The above-mentioned plasmids originating in the genomic library of theL22-8B strain were transformed into S. cerevisiae SH3328 by theabove-mentioned method of R. H. Schiestl. From the transformed strains,an aureobasidin resistant strain was isolated. Then a plasmid DNAcontaining the scaur2^(R) gene was recovered from this transformant bythe above-mentioned method of I. Hagan et al. This plasmid wastransformed into a yeast again and it was confirmed that thetransformant had acquired aureobasidin resistance. This plasmid, whichcontained a DNA of 8.5 kb, was named pSCAR2. FIG. 3 shows therestriction enzyme map of the DNA fragment of 8.5 kb containing thisaureobasidin resistant gene scaur2^(R) . E. coli HB101 having thisplasmid pSCAR2 introduced therein was named and designated asEscherichia coli HB101/pSCAR2. This strain has been deposited atNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology in accordance with the Budapest Treatyunder the accession number FERM BP-4484. By using BamHI, EcoRI, HindIIIand PstI, DNA fragments of various sizes were prepared and cloned intothe pWHS vector. These plasmids were transformed into S. cerevisiaeDKD5D in accordance with the above-mentioned method of R. H. Schiestl etal. Then it was examined whether these transformants had acquiredaureobasidin resistance or not. As a result, none of the transformantsof the DNA fragments was a resistant one. Thus it has been clarifiedthat the DNA fragment of the full length is necessary for the expressionof the aureobasidin resistance.

2-h) Isolation of aureobasidin sensitive gene scaur2^(S) correspondingto aureobasidin resistant gene scaur2^(R)

An E. coli stock containing the genomic library of Example 2-e) preparedfrom normal cells of S. cerevisiae DKD5D was spreaded on an LB agarmedium containing ampicillin and tetracycline and incubated at 37° C.overnight. The colonies thus formed were transferred onto a nylonmembrane (Hybond™-N) and the colony hybridization was performed. As aprobe the DNA fragment of 8.5 kb obtained in the above Example 2-g) andlabeled with [α-³²P] dCTP by using a random primer DNA labeling kit wasused. As the results of screening of 2×10⁴ colonies, several clonesbeing hybridizable with the probe were obtained. Some of these clonescontained a DNA fragment of 4.6 kb while others contained a DNA fragmentof 3.9 kb. From the restriction enzyme maps of these DNA fragments, itwas found out that these DNA fragments were each a part of thescaur2^(S) gene shown in FIG. 3. These DNA fragments were ligatedtogether to thereby give a scaur2^(S) fragments shown in FIG. 3. The DNAfragment of 8.5 kb thus obtained was subcloned into pUC 118 and then theDNA nucleotide sequence was determined (SEQ ID No. 23 in SequenceListing). Based on the nucleotide sequence of SEQ ID No. 23 in SequenceListing, the amino acid sequence represented by SEQ ID No. 24 inSequence Listing was estimated.

Example 3

Gene disruption test on spaur1^(S) and scaur1^(S) genes

3-a) Gene disruption test on spaur1^(S) gene

In order to examine whether the aureobasidin sensitive gene spaur1^(S)is necessary in the cell growth by the gene disruption test, the plasmidpUARS2R prepared in Example 1-d) was first cleaved with BalI andEcoT22I. After eliminating a DNA fragment of 240 bp, the residual DNAfragment was blunted by using a DNA blunting kit (manufactured by TakaraShuzo Co., Ltd.). Then this DNA was ligated with a DNA containing ura4⁺gene of 1.7 kb, which had been obtained by excising from a pUC8ura4plasmid [Mol. Gen. Genet., 215, 81-86 (1988)] by cleaving with HindIIIand blunting, to thereby give a plasmid pUARS2RBT22::ura4-1 and anotherplasmid pUARS2RBT22::ura4-6 in which the ura4 DNA had been inserted inthe opposite direction. Both of these disrupted genes were excised fromthe vector pUC118 by cleaving with SacI and HindIII andARS2RBT22::ura4-1 and ARS2RBT22::ura4-6 (FIG. 4), which were spaur1^(S)DNA fragments containing ura4⁺, were purified. The purified DNAfragments were transformed into diploid cells Schizo. pombe C525(h⁹⁰/h⁹⁰, ura4-D18/ura4-D18, leu1/leu1, ade6-M210/ade6-M216) by theabove-mentioned method of Okazaki et al. and then a transformant wasscreened on an SD agar plate containing leucine. In the transformantthus obtained, one of a pair of spaur1^(S) genes on the chromosome hadbeen is replaced by the disrupted gene ARS2RBT22::ura4-1 orARS2RBT22::ura4-6 introduced thereinto. These cells were allowed toundergo sporulation on a sporulation medium MEA and subjected to thetetrad analysis. As a result, it was found out that two of the fourascospores formed colonies but the residual two spores formed no colony.That is to say, the spores suffering from the replacement of the normalspaur1^(S) gene by the disrupted gene ARS2RBT22::ura4-1 were notpropagated. It has been thus revealed that the spaur1^(S) gene isessentially required for the growth of the cells.

3-b) Gene disruption test on scaur1^(S) gene

The plasmid pSCAR1 prepared in Example 2-e) was partially digested withHindIII to thereby give a DNA fragment of 3.5 kb shown in FIG. 2. ThisDNA fragment was cloned into the HindIII site of pUC119 and the obtainedproduct was named pSCAR3. The obtained pSCAR3 was cleaved with StuI andEcoT22I. After eliminating a DNA fragment of 0.3 kb, the obtained DNAwas ligated with a DNA fragment (1.1 kb) of URA3 gene which had beenobtained by cleaving a plasmid pYEUra3 (manufactured by ClontechLaboratories, Inc.) with HindIII and EcoRI and blunting. Thus, a plasmidpUSCAR3.ST22::URA3⁺ and another plasmid pUSCAR3.ST22::URA3A, in whichthe URA3 gene had been inserted in the opposite direction, wereobtained. These disrupted genes were excised in the EcoRI site in thescaur1^(S) gene and the EcoRI site in the pUC119 vector by cleaving withEcoRI. The scaur1^(S) DNA fragments containing URA3, SCAR3.ST22::URA3+and SCAR3.ST22::URA3A (FIG. 5), were purified. The purified DNAfragments were transformed into diploid cells of S. cerevisiae AOD1(mating type a/α, genotype ura3-52/ura3-52, leu2-3 112/leu2-3 112,trp1/TRP1, thr4/THR4, his4/HIS4) by the above-mentioned method of R. H.Schiestl and transformants were screened on an SD agar plate containingleucine. The transformants thus obtained were allowed to undergosporulation on a sporulation medium SP (1% of potassium acetate, 2% ofagar) and subjected to the tetrad analysis. As a result, it was foundout that two of the four ascospores underwent germination and formedcolonies but the residual two spores did not undergo colony formation.That is to say, the spores suffering from the replacement of thescaur1^(S) gene by the disrupted gene were not propagated. It has beenthus revealed that the scaur1^(S) gene is essentially required for thegrowth of the cells.

Example 4

Examination on the expression of aureobasidin sensitive gene spaur1 bynorthern hybridization

From a normal strain or a resistant strain of Schizo. pombe, the wholeRNAs were extracted and purified by the method of R. Jensen et al.[Proc. Natl. Acad. Sci. USA, 80, 3035-3039 (1983)]. Further, poly(A)⁺RNAwas purified by using Oligotex™-dT30 (manufactured by Takara Shuzo Co.,Ltd.). The purified poly(A)⁺RNA (2.5 μg) was separated by theelectrophoresis on a 1.2% agarose gel containing formaldehyde andtransferred onto a nylon membrane (Hybond™-N). After immobilizing, thehybridization was performed with the use of a HindIII-SacI fragment (2kb) of the spaur1^(R) gene labeled with [α-³²P]dCTP as a probe. As aresult, both of the normal cells and the resistant cells showed a bandof the same amount of about 2 kb. In both cases, this amount underwentno change in the logarithmic growth phase and the stationary phase (FIG.10). FIG. 10 is an autoradiogram showing the results of the northernhybridization wherein mRNAs obtained from cells of a sensitive strain ofSchizo. pombe in the logarithmic growth phase (lane 1), cells of aresistant strain in the logarithmic growth phase (lane 2), cells of thesensitive strain in the stationary phase (lane 3) and cells of theresistant strain in the stationary phase (lane 4) are electrophoresed ona 1.2% agarose gel containing formaldehyde.

Example 5

Determination of the activity of scaur1^(S) gene

5-a) Construction of plasmid YEpSCARW3 (FIG. 9) and YEpSCARW1

The plasmid pSCAR1 prepared in Example 2-e) was cleaved with HindIII anda fragment of 2 kb containing the whole ORF was excised. This fragmentwas inserted into the HindIII site of a expression-plasmid YEp52 havinga promoter Gal10, the expression of which was induced by galactose in amedium. The plasmid having the scaur1^(S) gene which had been insertedin such a direction as to be normally transcribed by the promoter Gal10was named YEpSCARW3. FIG. 9 shows the structure of this plasmid.Further, the plasmid having the scaur1^(S) gene inserted in the oppositedirection was named YEpSCARW1.

5-b) Transformation by plasmids YEpSCARW3 and YEpSCARW1

By using 5 μg portions of the plasmids YEpSCARW3 and YEpSCARW1, thediploid S. cerevisiae cells with the disrupted scaur1^(S) gene preparedin Example 3-b) were transformed. Then transformants were screened on anSD agar plate. These transformants were allowed to undergo sporulationon an SP medium and then subjected to the tetrad analysis. When theexpression of the scaur1^(S) gene was induced by using a YPGal medium(1% of yeast extract, 2% of polypeptone, 2% of galactose), theascospores formed from the diploid cells transformed by YEpSCARW3 allunderwent germination while two of the four ascospores formed from thediploid cells transformed by YEpSCARW1 underwent germination but not theremaining two. It is thus conceivable that the cells with the disruptedscaur1^(S) gene have reverted to the normal state by introducingYEpSCARW3 containing the scaur1^(S) gene into these cells. Accordingly,the use of these cells with the disrupted scaur1^(S) gene as a hostmakes it possible to determine the activity of normal aur1-analogousgenes carried by other organisms.

Example 6

Confirmation and cloning of aur1 and aur2 genes (caaur1, caaur2) carriedby C. albicans

6-a) Detection of aur1 gene by the PCR method

Poly(A)⁺RNA was extracted and purified from an aureobasidin sensitivestrain C. albicans TIMM0136 by the same method as the one employed inExample 4. By using the poly(A)⁺RNA (5 μg) as a template, adouble-stranded cDNA was synthesized on a cDNA synthesizing system Plus(manufactured by Amersham) with the use of an oligo(dT) primer. Mixedprimers for PCR corresponding to amino acid sequence regions beingcommon to the amino acid sequences of S. cerevisiae and Schizo. pombewere synthesized on a DNA synthesizer and purified. That is to say, aprimer of SEQ ID No. 25 in Sequence Listing corresponding to the regionof amino acids at the 184- to 192-positions of SEQ ID No. 18 in SequenceListing of Schizo. pombe (from the 184- to 192-positions of SEQ ID No.22 in Sequence Listing of S. cerevisiae) and another primer of SEQ IDNo. 26 in Sequence Listing corresponding to the region of amino acidsfrom the 289- to 298-positions of Schizo. pombe (from the 289- to298-positions of SEQ ID No. 22 in Sequence Listing of S. cerevisiae)were employed.

PCR was performed by using these primers and the above-mentioned cDNA asa template by repeating a cycle comprising treatment at 94° C. for 30seconds, one at 48° C. for 1 minute and one at 72° C. for 2 minutes 25times. As a result, a DNA (about 350 bp) being almost the same as S.cerevisiae and Schizo. pombe in length was amplified (FIG. 6). FIG. 6shows a pattern obtained by carrying out PCR with the use of cDNA of C.albicans (lane 1), cDNA of S. cerevisiae (lane 2) and cDNA of Schizo.pombe (lane 3) as a template, electrophoresing each PCR product on anagarose gel and staining with ethidium bromide.

6-b) Cloning of aur1 gene (caaur1) of C. albicans

(i) Genomic DNA was extracted and purified from a strain C. albicansTIMM0136 by the same method as the one described in Example 1-c). Afterpartially digesting with HindIII, the DNA fragment was ligated with aTraplex119 vector which had been completely digested with HindIII andtransformed into E. coli HB101. Thus a genomic library of C. albicanswas prepared. From this library, a DNA fragment of 4.5 kb containing theaur1 gene of C. albicans was cloned by using the DNA fragment of C.albicans obtained by the PCR described in Example 6-a), which had beenlabeled with [α-³²P]dCTP by using a random primer DNA labeling kit(manufactured by Takara Shuzo Co., Ltd.), as a probe. This DNA fragmenthad a restriction enzyme map shown in FIG. 7 and the DNA nucleotidesequence thereof is represented by SEQ ID No. 27 in Sequence Listing.Based on this nucleotide sequence, it was estimated that the caaur1 genecoded for a protein having the amino acid sequence represented by SEQ IDNo. 28 in Sequence Listing. When compared with the scaur1^(S) protein, ahomology of as high as 53% was observed. A Traplex119 vector having thiscaaur1 gene integrated therein was named pCAAR1, while E. coli HB101transformed by this plasmid was named and designated as Escherichia coliHB101/pCAAR1. This strain has been deposited at National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology in accordance with the Budapest Treaty under the accessionnumber FERM BP-4482.

Next, pCAAR1 was treated with HindIII to thereby give caaur1 of 4.5 kb.Further, it was integrated into pTV118 which had been completelydigested with HindIII to thereby prepare a plasmid for expressingcaaur1. This plasmid was named pTCAAR1.

(ii) Genomic DNA was extracted and parified from a strain C. albicansTIMM1768 [The Journal of Antibiotics, 46, 1414-1420(1993)] by the samemethod as the one described in Example 1-c). After partially digestingwith HindIII, the DNA fragment was ligated with a pUC118 vector whichhad been completely digested with HindIII and transformed into E. coliHB101. Thus a genomic library of C. albicans TIMM1768 was prepared. Fromthis library, a DNA fragment of 4.5 kb containing the aur1 gene of C.albicans TIMM1768 was cloned by the colony hybridization with the sameprobe as that described in Example 6-b)-(i). This DNA fragment had thesame restriction enzyme map as that shown in FIG. 7. Next, a part of theDNA sequence containing a ORF in this DNA fragment was determined. TheDNA nucleotide sequence thereof is represented by SEQ ID No. 35 inSequence Listing. Based on this nucleotide sequence, it was estimatedthat this gene coded for a protein having the amino acid sequencerepresented by SEQ ID No. 36 in Sequence Listing. When the amino acidsequence of the caaur1 protein C. albicans TIMM1768 was compared withthat of the caaur1 protein of C. albicans TIMM0136, the amino acidsequences of the 1- to 381-positions and the 383- to 423-positions andthe 425- to 471-positions of caaur1 protein (SEQ ID No. 28 in SequenceListing) in C. albicans TIMM0136 were identical with the amino acidsequences of the 2- to 382-positions and the 384- to 424-positions andthe 426- to 472-positions, respectively, of caaur1 protein (SEQ ID No.36 in Sequence Listing) in C. albicans TIMM1768.

However, serines at the 382- and 424-positions of SEQ ID No. 28 inSequence Listing were replaced with prolines at the 383- and425-positions of SEQ ID No. 36 in Sequence Listing.

6-c) Cloning of aur2 gene (caaur2) of C. albicans

Genomic DNA of a strain C. albicans TIMM0136 was digested with BamHI andligated with a pTV118 vector which had been completely digested withBamHI. Then it was transformed into E. coli HB101 to thereby prepare agenomic library of C. albicans. On the other hand, the DNA fragmentcontaining the scaur2^(S) gene obtained in Example 2-h) was cleaved withHindIII and PstI to thereby give a DNA fragment of 1.2 kb. This DNAfragment was labeled with [α-³²P]dCTP by using a random primer DNAlabeling kit. By using this labeled DNA fragment as a probe, theabove-mentioned C. albicans genomic library was screened by the colonyhybridization. Thus a plasmid containing a DNA fragment of 8.3 kb wasobtained. A part of the DNA sequence upstream of the BamHI site of thisDNA fragment was determined (SEQ ID No. 29 in Sequence Listing). Basedon this sequence, an amino acid sequence represented by SEQ ID No. 30 inSequence Listing was estimated. It corresponded to the amino acidsequence of the 1230- to 1309-positions of the amino acid sequence ofthe scaur2 gene (SEQ ID No. 24), having a homology of as high as 77%.Since this DNA fragment lacked a part of the C-end, the genomic libraryprepared in Example 6-b) was further screened by using this DNA fragmentas a probe. Thus a DNA fragment of 6.5 kb having the C-terminal part wasobtained. FIG. 8 shows the restriction enzyme map of the DNA regioncontaining the caaur2 gene thus clarified.

A pTV118 vector having the above-mentioned caaur2 gene of 8.3 kbintegrated therein was named pCAAR2N, while E. coli HB101 transformed bythis plasmid was named and designated as Escherichia coli HB101/pCAAR2N.This strain has been deposited at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology inaccordance with the Budapest Treaty under the accession number FERMBP4481.

Example 7

Preparation of antibody against protein coded for by scaur1^(S) gene andstaining of S. cerevisiae cells and detection of said protein by usingthis antibody

7-a) Preparation of antibody

SCAR1-1 (SEQ ID No. 33 in Sequence Listing) comprising a peptidecorresponding to the amino acids at the residue 103 to 113 in the aminoacid sequence of SEQ ID No. 22 in Sequence Listing having cysteine addedto the N-end thereof and SCAR1-2 (SEQ ID No. 34 in Sequence Listing)comprising a peptide corresponding to the amino acids at the residue 331to 348 in the amino acid sequence of SEQ ID No. 22 having cysteine addedto the N-end thereof were synthesized by the Fmoc solid phase synthesismethod and purified by reverse phase HPLC. Thus 10 mg portions of thesepeptides were obtained. To the N-terminal cysteine of each of thesesynthetic peptides, KLH was bound as a carrier protein. By using thisbinding product as an antigen, a rabbit was immunized and an antiserumwas obtained. This antiserum was further purified on an affinity columnprepared by binding the synthetic peptide employed as the antigen to anagarose gel. Thus a polyclonal antibody being specific for the syntheticpeptide was prepared.

7-b) Staining of S. cerevisiae cells with antibody

A strain S. cervisiae ATCC 9763 was cultured in a YNBG medium [0.67% ofyeast nitrogen base (manufactured by Difco), 2% of glucose] to therebygive a suspension of a concentration of 3×10⁷ cells/ml. To 1 ml of thiscell suspension were added 0.11 ml of a 1 M phosphate buffer (pH 6.5)and 0.17 ml of 37% formaldehyde. After slowly stirring at roomtemperature for 1 hour, the cells were harvested by centrifugation andthen suspended in 20 ml of an SS buffer (1 M of sorbitol, 0.2% ofβ-mercaptoethanol, 0.1 M phosphate buffer, pH 7.5) containing 20 μg/mlof Zymolyase 20T. After treating at 30° C. for 1 hour, the cells wereharvested, washed with the SS buffer, suspended in 1 ml of the SS buffercontaining 0.1% of Triton X-100 and then allowed to stand for 10minutes. This cell suspension was placed on a slide glass which had beencoated with poly(L-lysine) and allowed to stand for 10 minutes. Next, aPBS solution containing 1% of albumin (BSA) was dropped thereinto. Afterallowing to stand at room temperature for 15 minutes, the excessiveliquid was removed and then a PBS solution containing BSA containing0.02 mg/ml of the antiSCAR1-1 antibody was dropped thereinto. Afterallowing to stand at room temperature for 60 minutes and washing withPBS containing BSA three times, antirabbit IgG antibody labeled withFITC (antibody concentration 0.02 mg/ml) was layered over and allowed tostand at room temperature for 1 hour. After washing with a PBS solutioncontaining BSA, a small amount of a mounting solution, which was asolution prepared by dissolving 0.1 g of p-phenylenediamine in 10 ml ofCBS (150 mM of NaCl, 50 mM of CHES, pH 9.5), adjusting the pH value to9.0 with 10 N NaOH and further adding 90 ml of glycerol, was layeredover. Then a cover glass was placed thereon to thereby give a specimen.This specimen was observed under a fluorescence microscope to therebyexamine the intracellular distribution of the scaur1 protein. As aresult, it was found out that this protein was distributed all over thecells.

7-c) Detection of protein coded for by scaur1 gene by using antibody Theplasmid YEpSCARW3 prepared in Example 5-a) was introduced into a normalhaploid S. cerevisiae SH3328 to thereby give a transformant. Thistransformant was cultured in a YPGal medium or a YPD medium and thecells were harvested by centrifugation. The cells thus obtained weresuspended in a buffer (1% of Triton X-100, 1% of SDS, 20 mM of Tris-HCl,pH 7.9, 10 mM of EDTA, 1 mM of DTT, 1 mM of PMSF). Further, glass beadswere added thereto to disrupt the cells by vigorous vortex. Then an SDSloading solution was added thereto and the protein was denatured bytreating at 95° C. for 5 minutes. After centrifuging, a part of theobtained supernatant was subjected to SDS-PAGE and the protein thusseparated was transfered onto an Immobilon membrane (manufactured byMILLIPORE). This Immobilon membrane was treated with Block Ace(manufactured by Dainippon Pharmaceutical Co., Ltd.). Then theantiSCAR1-2 antibody prepared in 7-a) was reacted therewith as a primaryantibody. After washing, antirabbit IgG antibody labeled with peroxidasewas reacted therewith as a secondary antibody and the mixture wasthoroughly washed. Next, it was color-developed with diaminobenzidineand a band of the scaur1 protein was detected. FIG. 11 shows theresults.

FIG. 11 shows the results of the detection of the protein prepared fromthe cells incubated in the YPD medium (lane 1) and the protein preparedfrom the cells incubated in the YPGal medium (lane 2), each subjected toSDS-PAGE, by using the antiSCAR1-2 antibody. The cells incubated in theYPGal medium, of which scaur1 gene had been induced, showed a specificband.

Example 8

Construction of chromosome integration vector containing aureobasidinresistant gene

8-a) Construction of replication vector containing scaur1^(R)

A plasmid pSCAR1 was prepared from Escherichia coli HB101/pSCAR1 (FERMBP-4483) which carried a plasmid pSCAR1 containing scaur1^(S). Then theobtained plasmid was partially cleaved with HindIII and thus a DNA of3.5 kb containing scaur1^(S) was separated therefrom. This DNA (3.5 kb)was ligated to a vector pUC118 cleaved with HindIII to thereby prepare aplasmid pUscaur1^(S). This plasmid pUscaur1^(S) was transformed intoEscherichia coli CJ236 to thereby prepare ssDNA.

Next, a site-specific DNA mutation was introduced by using Mutan-K kit(manufactured by Takara Shuzo Co., Ltd.) with the use of a syntheticoligonucleotide for introducing mutation represented by SEQ ID No. 37 inthe Sequence Listing, which had been synthesized and purified, and theabove-mentioned ssDNA. That is to say, the use of the oligonucleotiderepresented by SEQ ID No. 37 in the Sequence Listing made it possible toobtain scaur1^(R) wherein the codon TTT of the 158th amino acid residuePhe in the ORF of the gene scaur1^(S) had been replaced by the codon TATof Tyr. This plasmid having a DNA coding for Aur1^(R)p (F158Y) wasdesignated as pUscaur1R.

8-b) Amplification of scaur1^(R) by PCR method

By using the plasmid pUscaur1^(R) which carried a HindIII fragment of3.5 kb containing scaur1^(R), scaur1^(R) (about 1.9 kb) was amplified bythe PCR method. Regarding primers employed herein, XhoI and KpnI sitesbad been designed in primers in order to clone the amplified scaur1^(R)into the plasmid vector pYES2 (manufactured by Invitrogen corporation)and thus primers represented by SEQ ID Nos. 38 and 39 in the SequenceListing were synthesized.

The reaction was effected in the following manner. 100 μl of a PCRsolution containing 28 μl of a PCR buffer [capable of giving finalconcentrations of 10 mM of S Tris-HCl (pH 8.3), 50 mM of KCl, 1.5 mM ofMgCl₂, 0.1 mM of dATP, 0.1 mM of dCTP, 0.1 mM of dTTP and 0.1 mM ofdGTP], 1 μl of 2.5 U of Ampli Taq DNA polymerase (Manufactured byPerkin-Elmer), 0.5 μl portions of 20 pmol of the primers represented bySEQ ID Nos. 38 and 39 in the Sequence Listing, 1 μl of the plasmid and69 μl of H₂O was maintained at an initial temperature of 94° C. for 1minute, and then heated successively at 94° C. for 1 minute, at 50° C.for 2 minutes and at 72° C. for 3 minutes. This heating cycle wasrepeated 35 times. Next, the reaction mixture was maintained at 72° C.for 10 minutes to thereby effect the amplification by PCR. Then the PCRamplification product was cleaved with KpnI and XhoI and electrophoresedon an agarose gel and the target DNA fragment of about 1.9 kb wasrecovered from the gel and purified by using Suprec™-01 (manufactured byTakara Shuzo Co., Ltd.).

8-c) DNA ligation and transformation

About 0.3 μg of the DNA fragment (about 1.9 kb) purified in the abovestep was ligated to about 0.1 μg of pYES2, which had been digested withXhoI and KpnI, by using a DNA ligation kit (manufactured by Takara ShuzoCo., Ltd.).

Next, 7 μl of the above-mentioned ligation mixture was added to 200 μlof competent cells of Escherichia coli HB101. These cells were allowedto stand on ice for 30 minutes, at 42° C. for 1 minute and then on iceagain for 1 minute. Then 800 μl of an SOC medium [Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989)] was added thereto. After incubating at 37° C. for 1 hour,these Escherichia coli cells were spread onto an L-broth agar mediumcontaining 50 μg/ml of ampicillin and incubated at 37° C. overnight.Thus a transformant was obtained.

This transformant was incubated in S ml of an L-broth medium containing50 μg/ml of ampicillin at 37° C. overnight. From this culture, a plasmidDNA was prepared in accordance with the alkali method (MolecularCloning, cited above). The plasmid thus obtained was named pYES2aur1.

8-d) Construction of chromosome integration vector

About 0.4 μg of the above-mentioned plasmid pYES2aur1 was digested withXbaI and KpnI and electrophoresed on an agarose gel. Then a DNA fragmentof about 1.9 kb containing scaur1^(R) was recovered from the gel andpurified by using Suprec-01.

Similarly, about 0.4 μg of the plasmid vector pUC19 was digested withSspI and PvuII and electrophoresed on an agarose gel. Then a DNAfragment (about 1.8 kb) containing an ampicillin resistant gene andColE1 origin was recovered from the gel and purified. About 0.1 μgportions of these DNA fragments thus purified were blunt-ended with theuse of a DNA blunting kit (manufactured by Takara Shuzo Co., Ltd.).Further, about 0.1 μg portions of these blunted DNA fragments weresubjected to a ligation reaction. The ligation reaction was effected byusing a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.).

Subsequently, the plasmid was integrated into Escherichia coli JM109.After incubating, a transformant and a plasmid DNA were prepared. Theplasmid thus obtained was named plasmid pAUR1. Next, this plasmid wascleaved with StuI to thereby prepare a chromosome integration vector.

Example 9

Construction of chromosome integration vector containing aureobasidinresistant gene

9-a) Amplification of DNA fragment of scaur1^(R) having mutationintroduced therein by PCR

In order to replace the 240th amino acid residue Ala of Aur1^(R)p(F158Y) by Cys, a primer, wherein the codon GCT of Ala had been changedinto the codon TGT of Cys, represented by SEQ ID No. 48 in the SequenceListing was synthesized and purified. By using the plasmid pUscaur1^(R)described in Example 8-a), which carried a HindIII fragment (3.5 kb)containing scaur1^(R) at the HindIII site of pUC119, as a template, wasamplified a DNA fragment (about 1.4 kb) which contained a sequence ofabout 500 bp coding for the amino acid sequence on the C-terminal sideof Aur1^(R)p (F158Y, A240C), wherein GCT had been changed into TGT, bythe PCR method with the use of the primer represented by SEQ ID No. 48in the Sequence Listing and a primer M13M4. The PCR was effected in thefollowing manner. To 28 μl of a PCR buffer [capable of giving finalconcentrations of 10 mM of Tris-HCl (pH 8.3), 50 mM of KCl, 1.5 mM ofMgCl₂, 0.1 mM of dATP, 0.1 mM of dCTP, 0.1 mM of dTTP and 0.1 mM ofdGTP] were added 2.5 U of Ampli Taq DNA polymerase, 100 pmol portions ofthe primer represented by SEQ ID No. 48 in the Sequence Listing and theprimer M13M4, 1 ng of pUscaur1^(R) and distilled water to thereby give100 μl of a PCR solution. This reaction mixture was then heatedsuccessively at 94° C. for 1 minute, at 55° C. for 1.5 minutes and at72° C. for 1.5 minutes. This cycle was repeated 30 times. Next, the PCRproduct was cleaved with SalI and SnaI and electrophoresed on an agarosegel. The target DNA fragment of about 1.3 kb was recovered from the geland purified.

9-b) Construction of plasmid containing DNA coding for Aur1^(R)p (F158Y,A240C)

pUscaur1^(R) was cleaved with SalI and SnaI and electrophoresed on anagarose gel. The target DNA fragment of 5.3 kb was recovered andpurified. To this DNA fragment was ligated the DNA fragment of 1.3 kbobtained in Example 9-a). The obtained plasmid, which had a DNA(scaur1^(R)-C) coding for Aur1^(R)p (F158Y, A240C), was namedpUscaur1^(R)-C. To effect transformation by integrating it into thechromosome of sake yeast, pUscaur1^(R)-C was linearized by cleaving withStuI prior to use. As a control, pUscaur1^(R) was also linearized withStuI.

9-c) Construction of plasmid containing DNA coding for Aur1^(R)p (A240C)pUscaur1^(S) was cleaved with SalI and SnaI and electrophoresed on anagarose gel. The target DNA fragment of 5.3 kb was recovered andpurified. This DNA fragment was ligated the DNA fragment of 1.3 kbobtained in Example 9-a). The obtained plasmid, which had a DNA codingfor Aur1^(R)p (A240C) wherein the 240th residue Ala of Aur1^(S)p hadbeen replaced by Cys, was named pUscaur1A240C. To effect transformationby integrating it into the chromosome of sake yeast, pUscaurA240C waslinearized by cleaving with StuI prior to use.

Example 10

Transformation by using sake yeast as host

10-a) About 10 μg of the linearized vector of the plasmid pAUR1described in Example 8 was introduced into Sake yeast Kyokai k-701 bythe lithium acetate method [Journal of Bacteriology, 153, 163 (1983)].

Namely, to Sake yeast Kyokai K-701, which had been suspended in a 0.1 Mlithium acetate solution (about 1.3×10⁸ cells/100 μl of 0.1 M lithiumacetate), was added 10 μg of the vector which had been prepared throughthe linearization of scaur1^(R) by cleaving with StuI at one position.After treating at 30° C. for 30 minutes and then at 42° C. for 15minutes, the cells were harvested by centrifugation and pre-incubated in5 ml of a YPD liquid medium. After pre-incubating in a YPD liquid mediumcontaining 0.4 μg/ml of aureobasidin A, transformants were obtained on aYPD agar medium containing 0.8 μg/ml of aureobasidin A. Thistransformant was named Sake yeast Kyokai K-701/pAUR1.

This transformant was subcultured over three generations in the absenceof aureobasidin A and then the sensitivity to aureobasidin was assayed.As a result, it showed eight times as much aureobasidin resistance (MIC1.56 μg/ml) as that of the parent strain (i.e., Sake yeast KyokaiK-701), which indicated that the aureobasidin resistance was sustained.Thus it has been confirmed that the aureobasidin resistance introducedon the host chromosome is usable as a selective marker.

10-b) To compare the activities of pUscaur1^(R)-C prepared in Example9-b), pUscaur1A240C prepared in Example 9-c) and pUscaur1^(R) preparedin Example 8-a), 5 μg of the plasmid, which had been linearized withStuI, was introduced into Sake yeast Kyokai K-701 by the lithium acetatemethod. Namely, to sake yeast, which had been suspended in a 0.1 Mlithium acetate solution (pH 7.5) and made competent, were added 5 μg ofthe plasmid, which had been linearized by cleaving with StuI at oneposition, and 850 μl of 40% polyethylene glycol/0.1 M lithium acetate.After treating at 30° C. for 30 minutes and then maintaining at 42° C.for 15 minutes, the cells were harvested, pre-incubated in 5 ml of a YPDliquid medium for 1 hour or overnight and then smeared on a YPD agarmedium containing aureobasidin A at various concentration. Afterincubating at 30° C. for 3 to 4 days, transformants having a resistanceto aureobasidin A were obtained. As Table 4 shows, the transformantprepared by using the StuI-linearized pUscaur1^(R)-C could grow even inthe medium containing 5 μg/ml of aureobasidin A. These transformantssustained an aureobasidin A resistance at least 10 times higher thanthat of the parent strain even after being subcultured over severalgenerations. The transformant obtained by using the linearizedpUscaur1^(R)-C showed an MIC to aureobasidin A of 20 μg/ml or above.Thus it has been confirmed that the Aur1^(R)p (F158Y, A240C) is usableas an effective selective marker for sake yeast. As Table 4 shows, theStuI-linearized pUscaur1A240C exceeded the StuI-linearized pUscaur1^(R)in the activity of imparting resistance. That is to say, the mutation atthe 240th residue Ala resulted in the expression of the activity ofimparting a stronger resistance.

TABLE 4 Pre- No. of transformants/μg DNA incubation Aureobasidin Aconcn. (μg/ml) Plasmid time 0.5 1.0 5.0 StuI-linearized 1 hour  0   0 0pUscaur1R overnight  5   4 0 StuI-linearized 1 hour 170  73 0pUscaur1R-C overnight 2368  2024 64  StuI-linearized 1 hour  18   1 0pUscaur1A240C overnight 160  152 0 no plasmid 1 hour  0   0 0 (control)overnight  0   0 0

Example 11

Transformation by chromosome integration vector having aureobasidinresistant gene and AARE gene

11-a) Construction of plasmid containing AARE gene

0.5 μg of the plasmid pAUR1 was cleaved with SphI and electrophoresed onan agarose gel. Then the linearized plasmid pAUR1 was recovered from thegel and purified.

Next, a plasmid pYHA201 was prepared from a yeast BJ2168, which carrieda plasmid pYHA201 containing the AARE gene described in Japanese PatentLaid-Open No. 254680/1991 (i.e., Saccharomyces cerevisiaeBJ2168/pYHA201; FERM P-11570). 0.5 μg of this plasmid pYHA201 wasdigested with SphI and the DNA fragments (about 3.2 kb) were isolatedand purified.

This DNA fragment of about 3.2 kb contained the AARE gene bound to thedownstream of the ADHI promotor.

By using each 0.2 μg portions of both the purified SphI-cleaved DNAfragments, a ligation reaction was effected by using a DNA ligation kit(manufactured by Takara Shuzo Co., Ltd.). Subsequently, the plasmid wasintegrated into Escherichia coli JM109 and incubated to thereby give atransformant and a plasmid DNA. The plasmid thus obtained was namedplasmid pAUR1aare, while the transformant thus obtained was named andindicated as Escherichia coli JM109/pAUR1aare and has been deposited atNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology under the accession number FERMP-14366.

11-b) Expression of AARE in sake yeast

By using about 10 μg of the above-mentioned plasmid pAUR1aare, theplasmid pAUR1aare linearized was transformed into Sake yeast KyokaiK-701 in the same manner as the one described in Example 10. Then anaureobasidin resistant transformant was obtained on a YPD agar mediumcontaining 0.4 μg/ml of aureobasidin A.

The transformant thus obtained was named and indicated as Saccharomycescerevisiae K701/pAUR1aare and has been deposited at National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology under the accession number FERM P-14379.

Next, this transformant was inoculated into 5 ml of a minimal medium[0.67% of Bacto Yeast Nitrogen Base w/o Amino Acid (manufactured byDifco), 2% of glucose] and incubated at 30° C. for two days and then thecells were harvested by centrifugation.

After discarding the supernatant, the cells were washed with 2 ml ofwater and harvested again by centrifugation.

Subsequently, the cells were suspended in 700 μl of a 0.2 M sodiumphosphate buffer Solution (pH 7.2).

To this suspension were added 400 μl of glass beads (0.40 to 0.60 mm indiameter) and the cells were disrupted by vigorously stirring underice-cooling.

After centrifuging, the supernatant was recovered and regarded as aextract.

Also, extracts of Sake yeast Kyokai K-701 and Sake yeast KyokaiK-701/pAUR1 obtained in Example 10 were prepared in the same manner.

The acylamino acid releasing enzyme activity of each of the extractsthus obtained was measured by the following method. 0.89 ml of a 0.5%dimethylformamide-0.2 M sodium phosphate buffer solution (pH 7.2)containing 0.020 mM of an amide prepared from N-acetyl-L-methionine and7-amino-4-methylcoumarin (AMC) was preheated at 37° C. for about 5minutes. Then 100 μl of the above-mentioned extract was added theretoand the resulting mixture was incubated at 37° C. for 15 minutes. Afterthe completion of the reaction, 10 μl of 10% SDS was added to therebycease the reaction and the intensity of fluorescence was measured with afluorophotometer. Namely, the excitation wavelength and the measurementwavelength were set respectively to 380 nm and 440 nm. The amount of theliberated AMC was determined by preparing a standard curve with the useof AMC samples of known concentrations and comparing the obtained datatherewith.

100 μl of the extract of S. cerevisiae K701/pAUR1aare had an activity ofliberating about 25 pmol of AMC in 15 minutes in the above reactionsystem.

On the other hand, the extract of Sake yeast Kyokai K-701 having noplasmid and the extract of Sake yeast Kyokai K-701/pAUR1 obtained inExample 10 showed each no AARE activity.

Further, an analysis was effected by the southern hybridization with theuse of the aureobasidin resistant gene as a probe. As a probe in thehybridization, use was made of an scaur1^(R) fragment (about 1.6 kb)which had been amplified by the PCR method with the use of the plasmidpAUR1 as a template and the primers represented by SEQ ID Nos. 40 and 41in the Sequence Listing. 100 ng of the fragment thus obtained waslabeled with [³²P]dCTP by using a BcaBEST™ labeling kit. The genomicDNAs of Sake yeast Kyokai K-701 and Saccharomyces cerevisiaeK701/pAUR1aare were cleaved with various restriction enzymes (HpaIhaving no cleavage site on pAUR1aare, BamHI having two cleavage sites onpAUR1aare), electrophoresed on an agarose gel and transferred onto thehybridization filter.

In FIG. 14, the lanes 1 and 2 show the results obtained by cleaving thegenomic DNA of Sake yeast Kyokai K-701 with HpaI (lane 1) or BamHI (lane3) and subjected to southern hybridization, while the lanes 2 and 4 showthe results obtained by cleaving the genomic DNA of Saccharomycescerevisiae K701/pAUR1aare with HpaI (lane 2) or BamHI (lane 4) andsubjected to southern hybridization. Since HpaI had no cleaving site onthe plasmid pAUR1aare but cleaved exclusively the genomic DNA, it wasproved from the lanes 1 and 2 that the aureobasidin resistant gene hadbeen integrated into one of a pair of chromosomes.

It was confirmed from the lanes 3 and 4 that the aureobasidin resistantgene had been homologously integrated into the aureobasidin sensitivegene on the chromosome of the sake yeast.

These results indicate that genes of the sake yeast were not disruptedby the random integration of the aureobasidin resistant gene.

As described above, by using the chromosome integration vector of thepresent invention, the resistance could be imparted to an aureobasidinsensitive fungi and, furthermore, a foreign gene could be expressed.

Example 12

Construction of recombinant plasmid containing aureobasidin resistantgene

12-a) Construction of pYC vector carrying DNA coding for Aur1^(R)p(F158Y, A240C)

By employing pUscaur1^(R)-C as a template, PCR was effected with the useof primers represented by SEQ ID Nos. 49 and 59 in the Sequence Listing.The PCR product (about 2.2 kb), which contained the DNA coding for theamplified Aur1^(R)p (F158Y, A240C), was cleaved with XhoI and KpnI andblunt-ended with the use of a blunting kit (manufactured by Takara ShuzoCo., Ltd.).

A pYC vector pYEUra3 (manufactured by Clontech Laboratories, Inc.) wascleaved with EcoRI and BamHI and blunt-ended with the use of a bluntingkit. Then it was ligated to the blunt-ended PCR product (about 2.2 kb)described above. The plasmid thus obtained was named pYCscaur1^(R)-C. Byemploying pUscaur1^(R) as a template, PCR was effected in the samemanner with the use of primers represented by SEQ ID Nos. 49 and 50 inthe Sequence Listing. The PCR product (about 2.2 kb), which containedthe DNA coding for the amplified Aur1^(R)p (F158Y), was cleaved withXhoI and KpnI and blunt-ended with the use of a blunting kit(manufactured by Takara Shuzo Co., Ltd.). A pYEUra3 was cleaved withEcoRI and BamHI and blunt-ended with the use of a blunting kit. Then itwas ligated to the blunt-ended PCR product (about 2.2 kb) describedabove. The plasmid thus obtained was named pYCscaur1R.

12-b) Construction of pYE vector carrying DNA coding for Aur1^(R)p(F158Y, A240C)

By using pUscaur1^(R)-C as a template, the DNA fragment (about 2.2 kb),which contained the DNA coding for Aur1^(R)p (F158Y, A240C), wasamplified by the PCR method with the use of a primer represented by SEQID No. 49 in the Sequence Listing and a primer M13M4. The PCR productwas cleaved with BamHI and electrophoresed on an agarose gel. Then thetarget DNA fragment (about 1.8 kb) was recovered from the gel andpurified. The plasmid pSCAR1 was cleaved with BamHI and thus a DNAfragment of 11.7 kb, from which a DNA fragment of 2.8 kb containingscaur1^(S) had been deleted, was obtained. This DNA fragment of 11.7 kbwas ligated to the above-mentioned DNA fragment of 1.8 kb containing theDNA coding for Aur1^(R)p (F158Y, A240C). Then a plasmid having thefragment of 1.8 kb inserted thereinto in the desired direction wasselected. This plasmid containing the DNA coding for Aur1^(R)p (F158Y,A240C) was named pWscaur1^(R)-C and employed in the transformation of ayeast for laboratory use.

Example 13

Transformation with the use of yeast as host

13-a) Transformation of sake yeast by pYCscaur1^(R)-C

By using 5 μg of a pYC plasmid pYCscaur1^(R)-C, Sake yeast Kyokai K-701was transformed by the lithium acetate method described in Example 8-a).As Table 5 shows, the obtained results are similar to those obtained inthe cases of the linearized plasmids. These transformants sustained eachan aureobasidin A resistance at least 10 times higher than that of theparent strain even after being subcultured over several generations.Thus it has been confirmed that Aur1^(R)p (F158Y, A240C) is usable as aneffective selective marker for sake yeast in the transformation by areplication vector.

TABLE 5 Pre- No. of transformants/μg DNA incubation Aureobasidin Aconcn. (μg/ml) Plasmid time 0.5 1.0 2.0 5.0 pYCscaur1R 1 hour  0  0  0 0 overnight  24  21  0  0 pYCscaur1^(R)-C 1 hour 386 172  18  1overnight 4824  4792  1692  136

13-b) Transformation of laboratory yeast by pWscaur1^(R)-C

By using a monoploid yeast DKD-5D for laboratory use (a, his3, trp1,leu2-3, 112) as a host, 5 μg of pWscaur1^(R)-C was transformed by thelithium acetate method described in Example 10-a).

For comparison, a transformant was screened on a minimal medium by usingan auxotrophic marker contained in the plasmid. As Table 6 shows, it hasbeen confirmed that Aur1^(R)p (F158Y, A240C) is usable as a selectivemarker which is comparable to the conventional auxotrophic markers in amonoploid yeast.

TABLE 6 Pre- No. of transformants/μg DNA incubation Minimal AureobasidinA concn. (μg/ml) Plasmid time medium 0.5 1.0 5.0 pWCscaur1^(R)-C 1 hour 192  248  181  58 overnight 2500 2780 2524 2044

Example 14

Transformation by using C. albicans as host

14-a) 5 μg of a linear plasmid, which had been prepared by cleavingpUscaur1^(R)-C with SalI capable of cleaving at one point in the pUC119region, was transformed into C. albicans TIMM0136 by the lithium acetatemethod. After the completion of the transformation, the cells wereincubated in a YPD medium for 1 hour or overnight and then smeared on aYPD agar medium containing aureobasidin A. As Table 7 shows,transformants having the resistance to aureobasidin were obtained byeffecting the pre-incubation overnight. These transformants showed eachan MIC of 10 μg/ml or above.

TABLE 7 No. of transformants/μg DNA Pre-incubation Aureobasidin A concn.(μg/ml) Plasmid time 0.25 1.0 SalI-linearized 1 hour  0 0 pUscaur1^(R)-Covernight 180 0

Example 15

Cloning of gene anaur1 regulating aureobasidin sensitivity andoriginating in A. nidulans

15-a) Isolation of aureobasidin resistant mutant of A. nidulans

A strain A. nidulans FGSC89 showing a sensitivity to aureobasidin at 5μg/ml was inoculated into an SD slant (containing 1% of polypeptone S,2% of glucose and 2% of agar) and incubated therein at 30° C. for 7days. After suspending in S ml of a 0.1% Tween 80 solution containing0.8% of NaCl, the suspension was filtered through a glass filter (3G3type) and the obtained filtrate was used as a conidium suspension. Thisconidium suspension was UV-radiated for 5 minutes, thus effectingmutagenesis. Under these conditions, the rate of survival was about 25%.After blocking off the light for 30 minutes or longer, the conidia wereinoculated into an SD plate and incubated at 30° C. for 4 days. Theconidia thus formed were collected with a glass filter, furtherinoculated into Cz+bi medium [containing 4.9% of Czapek solution agar(manufactured by Difco), 200 μg/ml of arginine and 0.02 μg/ml of biotin]containing 5 μg/ml of aureobasidin and incubated therein at 30° C. After2 or 3 days, eight aureobasidin resistant colonies were obtained.Although these cells showed a resistance to 80 μg/ml of aureobasidin,they were the same as the parent strain in the sensitivities toamphotericin B, cycloheximide and clotrimazole. Thus it was estimatedthat the resistance thus acquired was not a multiple drug resistance butone specific to aureobasidin.

15-b) Preparation of genomic library of aureobasidin resistant strain

From a strain R1 showing a particularly high resistance from among theaureobasidin resistant strains, genomic DNAs were extracted and purifiedin the following manner. After incubating in a PD medium [containing2.4% of potato dextrose broth (manufactured by Difco)] under shaking at30° C. for 2 days, the hyphae were collected with a glass filter (3G1type) and washed with distilled water. Then the cells were dehydratedand suspended in 20 ml of a protoplast generation solution [containing20 μg/ml of Yatalase (manufactured by Ozeki Shuzo), 0.8 M of NaCl and 10mM of a sodium phosphate buffer, pH 6.0]. Then the suspension was slowlystirred at 30° C overnight to thereby generate protoplasts. Thesuspension was filtered through a glass filter (3G2 type) and thus theprotoplasts were collected into the filtrate and then harvested bycentrifuging at 2,000 rpm for 5 minutes. After washing with 0.8 M NaCltwice, the protoplasts were suspended in 2 ml of a TE solution(containing 10 mM of Tris-HCl and 1 mM of EDTA, pH 8.0) and 2 ml of alysis solution (containing 2% of SDS, 0.1 M of NaCl, 10 mM of EDTA and50 mM of Tris-HCl, pH 7.0) was added thereto. After slowly stirring, themixture was maintained at room temperature for 15 minutes and thencentrifuged at 3,500 rpm for 10 minutes followed by the recovery of thesupernatant. Then an equivalent amount of a mixture ofphenol/chloroform/isoamyl alcohol (25/24/1) was added thereto and themixture in the tube was gently mixed and centrifuged at 3,000 rpm for 5minutes followed by the recovery of the upper liquid layer. Next, 2.5times by volume as much ethanol at −20° C. was added thereto. Theresulting mixture was allowed to stand at −80° C. for 10 minutes andthen centrifuged at 3,500 rpm for 15 minutes. The DNAs thus precipitatedwere dried. Then 0.5 ml of a TE solution and 2.5 μl of an RNase Asolution (20 μg/ml) were added thereto and the mixture was maintained at37° C. for 30 minutes.

After adding 0.5 ml of phenol/chloroform/isoamyl alcohol, the obtainedmixture was gently mixed and centrifuged at 10,000 rpm for 5 minutesfollowed by the recovery of the upper layer. This procedure was repeatedonce. After adding 0.5 ml of chloroform/isoamyl alcohol (24/1), theobtained mixture was gently mixed and centrifuged at 10,000 rpm for 5minutes followed by the recovery of the supernatant. Then 0.05 ml of 5 MNaCl and 0.5 ml of isopropyl alcohol were added and the mixture wasallowed to stand at −80° C. for 10 minutes and centrifuged at 10,000 rpmfor 15 minutes to thereby collect DNAs.

Eight μg of the genomic DNAs thus purified were partially digested bytreating with 4 U of a restriction enzyme BamHI at 37° C. for 15minutes. After deprotenization with phenol/chloroform, the DNA wasrecovered by ethanol precipitation. The DNAs were electrophoresed on a0.8% agarose gel and DNAs in a region of from 3 to 15 kb were extractedand purified. The DNAs thus obtained were ligated to a vector pDHG25[Gene, 98, 61-67 (1991)], which had been completely digested with BamHI,with the use of a DNA ligation kit (manufactured by Takara Shuzo Co.,Ltd.). Then Escherichia coli, HB101 was transformed thereby so as toprepare a genomic library of the resistant strain. The E. coli cellscontaining this genomic library were cultured in 50 ml of an LB medium(containing 1% of bactotrypton, 0.5% of bacto yeast extract and 0.5% ofsodium chloride) containing 100 μg/ml of ampicillin at 37° C. overnight.Next, plasmids were recovered and purified from the E. coli cells.

15-c) Expression and cloning of aureobasidin resistant gene anaur1^(R)

The plasmid originating in the genomic library of the aureobasidinresistant strain thus obtained was transformed into a strain A. nidulansFGSC89 by the following method. Namely, A. nidulans was incubated in aPD medium under shaking at 30C for 2 days. Then the hyphae werecollected by filtering the culture broth through a glass filter (3G1type) and washed with sterilized water. After sufficiently dehydrating,the cells were suspended in 10 ml of a protoplast generation solution.After reacting by slowly shaking at 30° C. for about 3 hours, the cellsuspension was filtered through a glass filter 3G3. Then the filtratewas centrifuged at 2,000 rpm for 5 minutes to thereby collect theprotoplasts therein. The collected protoplasts were washed with 0.8 MNaCl twice and suspended in Sol 1 (containing 0.8 M of NaCl, 10 mM ofCaCl₂ and 10 mM of Tris-HCl, pH 8.0) in such a manner as to give aprotoplast concentration of 2×10⁸/ml. Then 0.2 time by volume as muchSol 2 [containing 40% (w/v) of PEG4000, 50 mM of CaCl₂ and 50 mM ofTris-HCl, pH 8.0] was added thereto and well mixed.

10 μg of the plasmid originating in the genomic library was added to a0.2 ml portion of the protoplast suspension. After mixing well, themixture was allowed to stand in ice for 30 minutes and then 1 ml of Sol2 was added thereto. After mixing well, the mixture was allowed to standat room temperature for 15 minutes and then 8.5 ml of Sol 1 was addedthereto. After mixing well, the mixture was centrifuged at 2,000 rpm for5 minutes to thereby collect the protoplasts. 0.2 ml of Sol 1 was addedthereto and the resulting mixture was placed on the center of a minimummedium plate (containing 4.9% of Czapek solution agar, 0.8 M of NaCl and0.02 μg/ml of biotin) containing 5 μg/ml of aureobasidin. Next, 5 ml ofa soft agar medium (containing 3.5% of Czapek-Dox broth, 0.8 M of NaCl,0.02 μg/ml of biotin and 0.5% of agar) was layered thereon followed byincubation at 30° C. for 3 to 5 days. It was considered that thecolonies growing on this plate carried a plasmid containing anaureobasidin resistant gene.

Thus, about 70 colonies were formed on the aureobasidin-containingmedium. These colonies were transplanted into 20 ml of a Cz+Bi mediumand incubated at 30° C. for 2 days. Then DNA was recovered and purifiedfrom the cells thus propagated in accordance with the method for theextraction and purification of DNA described in Example 15-b). A strainE. coli HB101 was transformed by this DNA and spread on an LB platecontaining 100 μg/ml of ampicillin. Then a plasmid DNA was prepared fromthe E. coli colonies thus formed. This plasmid contained a DNA of 12 kband was named pR1-1. FIG. 19 shows the restriction enzyme map of the 12kb DNA contained in pR1-1. To further specify the resistant gene region,the DNA fragment of 12 kb was digested into fragments of various sizeswith restriction enzymes. Next, these fragments were cloned into avector pDHG25. Plasmids containing various DNAs were transformed into astrain A. nidulans FGSC89 so as to confirm whether the aureobasidinresistance could be thus acquired or not. As a result, it was revealedthat the activity of imparting aureobasidin resistance resided in afragment Bgl II (5.8 kb). Thus it was clarified that the gene anaur1^(R)was located in this fragment. FIG. 15 shows the restriction enzyme mapof this DNA fragment containing the aureobasidin resistant geneanaur1^(R) . This fragment was subcloned into a vector pUC118 and theobtained plasmid was named pUR1. By using this plasmid, the DNA sequenceof the DNA was identified. SEQ ID NO. 1 in the Sequence Listing showsthis base sequence. As this DNA sequence indicates, the gene anaur1^(R)is one composed of two exon regions containing an intron. It has beenrevealed that this gene encodes a protein having the amino acid sequencerepresented by SEQ ID NO. 2 in the Sequence Listing.

15-d) Cloning of aureobasidin sensitive gene anaur1^(S)

To obtain the cDNA of the aureobasidin sensitive gene from normal cellsof A. nidulans, total RNAs were first extracted from a strain A.nidulans FGSC89. Namely, this strain was incubated in 200 ml of a PDmedium and the cells were collected with the use of a glass filter (3G1type). After sufficiently dehydrating, the cells were quickly frozenwith liquid nitrogen. Then the frozen cells were powdered in a mortarand total RNAs (2.6 mg) were extracted and purified with the use of anRNA extraction kit (manufactured by Pharmacia). From 1 mg of these RNAs,12.8 μg of poly(A)⁺RNAs were prepared by using Oligotex-dT30<Super>(manufactured by Takara Shuzo Co., Ltd.). By using 5 μg of thepoly(A)⁺RNAs, cDNAs were synthesized with the use of a Takara cDNAsynthesizing kit (manufactured by Takara Shuzo Co., Ltd.). The cDNAsthus synthesized were ligated to a λ phage vector λSHlox™ (manufacturedby Novagen, Inc.) and subjected to in vitro packaging with the use ofPhage Maker™ System, Phage Pack Extract (manufactured by Novagen, Inc.)to thereby construct a cDNA library. This cDNA library was infected in ahost strain E. coli ER1647. After mixing with top agarose (an LB mediumcontaining 0.7% of agarose), it was layered on an LB plate and incubatedat 37° C. overnight to thereby form plaques. The plaques thus formedwere transferred onto a nylon membrane (Hybond-N, manufactured byAmersham) and subjected to plaque hybridization. As a probe, use wasmade of a DNA fragment of 2.6 kb obtained by cleaving the plasmid pUR1obtained in Example 15-c) with PstI and SalI. This DNA fragment waslabeled with [α-³²p]dCTP by using a random primer DNA labeling kit(manufactured by Takara Shuzo Co., Ltd.) and employed as a probe in thehybridization. As the result of screening of 4×10⁵ plaques, 8 phageclones hybridizable with the probe were obtained. Next, these phageswere subjected to automatic subcloning in E. coli to thereby give E.coli strains having plasmids wherein a cDNA-containing region had beenautomatically subcloned. The plasmids were purified from these strainsand the cDNAs were compared in length. Thus pSl5 having the longest cDNA(2.9 kb) was selected and subcloned into pUC118 followed by theidentification of the DNA sequence. This plasmid was named pANAR1. An E.coli strain transformed by pANAR1 was named Escherichia coliJM109/pANAR1 and has been deposited at National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology underthe accession number FERM BP-5180. FIG. 16 shows the restriction enzymemap of this cDNA. The DNA base sequence thereof is represented by SEQ IDNO. 3 in the Sequence Listing. As this base sequence indicates, the geneanaur1^(S) encodes a protein having the amino acid sequence representedby SEQ ID NO. 4 in the Sequence Listing. A comparison with the resistantgene anaur1^(R) has revealed that the base G at the position 1218 in SEQID NO. 3 has been mutated into T and, at the amino acid level, the aminoacid glycine at the position 275 has been converted into valine. It hasbeen further clarified that the genomic DNA has one intron (56 bp). FIG.19 shows the relation between the genomic DNA and cDNA.

Example 16

Confirmation and cloning of gene afaur1^(S) carried by A. fumigatus

16-a) Detection of gene afaur1^(S) by Northern hybridization

From a strain A. fumigatus TIMM1776, poly(A)⁺RNAs were extracted andpurified by the same method as the one of Example 15-d). Thepoly(A)⁺RNAs (1 μg) of A. fumigatus and A. nidulans were separated byelectrophoresing on a 1.2% agarose gel containing formaldehyde andtransferred onto a nylon membrane. After fixing, hybridization waseffected with the use of a HindIII fragment (741 bp) of the cDNA of thegene anaur1^(S) labeled with [α-³²p] dCTP as a probe. After hybridizingat 60° C. overnight, the mixture was washed at 60° C. with 0.5×SSC and0.1% SDS. In FIG. 19, the lanes 1 and 2 show the results of thehybridization of the poly(A)⁺RNAs obtained from A. nidulans and A.fumigatus respectively. As FIG. 20 clearly shows, autoradiography of thehybridization revealed that A. fumigatus and A. nidulans both had theaureobasidin sensitive genes of the same size. However, the band of A.fumigatus was very weak, which indicates that the homology between thesegenes is not so high.

16-b) Cloning of gene afaur1^(s) carried by A. fumigatus

By using the poly(A)⁺RNAs of A. fumigatus purified in Example 16-a), acDNA library of A. fumigatus was prepared in accordance with the methodfor the preparation of a cDNA library described in Example 15-d). Theabove-mentioned library was screened under the same hybridizationconditions as those of Example 16-a) with the use of a PstI-EcoRIfragment (921 bp) of the cDNA of A. nidulans as a probe. Thus eightphage clones were obtained. From these phase clone, the cDNA wasrecovered in the form of a plasmid by the method described in Example15-d). The plasmids were purified and the cDNAs were compared in length.Thus the plasmid having the longest cDNA (2.9 kb) among them wasselected. This cDNA was subcloned into pUC118 and the DNA sequence wasidentified. This base sequence, which is represented by SEQ ID NO. 12 inthe Sequence Listing, indicates that this gene encodes a protein havingthe amino acid sequence represented by SEQ ID NO. 5 in the SequenceListing. A comparison between the amino acid sequence of the afaur1^(s)protein of the strain A. fumigatus TIMM1776 with that of the anaur1^(s)protein of the strain A. nidulans FGSC 89 indicated that they had a highhomology (87%).

Example 17

Confirmation of genes regulating aureobasidin sensitivity carried by A.niger and A. oryzae

Genomic DNAs were extracted and purified from strains A. fumigatusTIMM1776, A. niger FGSC805 and A. oryzae IF05710 in accordance with themethod described in Example 15-b). Further, genomic DNAs were extractedand purified from yeast strains S. cerevisiae DKD-5D and Schizo. pombeJY745 in accordance with the method of P. Philippsen et al. [Methods inEnzymology, 194, 169-175 (1991)]. 5 μg portions of the genomic DNAs ofA. nidulans, A. fumigatus, A. niger, A. oryzae, S. cerevisiae andSchizo. pombe were cleaved with a restriction enzyme PstI, separated byelectrophoresing on a 0.8% agarose gel, transferred onto a nylonmembrane and fixed. Next, Southern hybridization was efected by using aPstI-EcoRI fragment of the cDNA of the anaur1 gene labeled with[α-³²p]dCTP as a probe. The hybridization was effected under the sameconditions as those described in Example 16-a). FIG. 21 shows theautoradiogram of the hybridization. As FIG. 21 clearly shows, genesregulating aureobasidin sensitivity occur in A. niger and A. oryzae too.It has been also revealed that the DNA of A. nidulans is nothybridizable with the aureobasidin sensitive genes of the yeasts S.cerevisiae and Schizo. pombe. In FIG. 21, the lanes 1, 2, 3, 4, 5 and 6show the results of the Southern hybridization of the genomic DNAs of A.nidulans, A. fumigatus, A. niger, A. oryzae, S. cerevisiae and Schizo.pombe respectively.

According to the present invention, a novel protein regulatingaureobasidin sensitivity and a gene coding for the protein, i.e., a generegulating aureobasidin sensitivity are provided. These substances areuseful in the diagnosis and treatment for diseases caused by organismshaving the above-mentioned gene, such as mycoses. The present inventionfurther provides an antisense DNA and an antisense RNA of this gene, anucleic acid probe being hybridizable with the gene, a process fordetecting the gene by using this nucleic acid probe, a process forproducing a protein regulating aureobasidin sensitivity by using atransformant having the gene introduced thereinto, an antibody for theprotein and a process for detecting the protein by using this antibody.They are also useful in the diagnosis and treatment of diseasesincluding mycoses.

In addition, the present invention provides a chromosome integrationvector with the use of a resistance to aureobasidin as a selectivemarker, which is useful in genetic recombination of fungi (inparticular, industrial fungi), a process for producing a transformanthaving this vector introduced thereinto, and a transformant obtained bythis process. They are effectively applicable to, for example thecreation of a transformant for producing a useful protein and breeding.The present invention also provides a protein capable of imparting astrong resistance to aureobasidin and a DNA coding for this protein.They imparted a resistance to an organism having an aureobasidinsensitivity and are useful as a selective marker for screening theorganism thus acquiring the resistance. They are highly usefulparticularly in, for example, the genetic engineering breeding of apractically usable yeast and the analysis of genetic information of C.albicans. In particular, an aureobasidin resistant gene originating inS. cerevisiae is highly useful in the breeding of S. cerevisiae and thepreparation of a transformant for producing a useful protein, because S.cerevisiae is a yeast which is not only widely employed as an industrialyeast but also highly safe in genetic recombination.

Both the aureobasidin-resistant genes obtained from yeasts and that frommold are isolated from the resistant mutant derived from yeasts andmold, respectively. And the aureobasidin-resistant gene of mold havefunction, giving an aureobasidin-resistance, identical to the genes ofyeasts. That is, these genes are functional homologs. Although aminoacid sequence of aureobasidin-resistant genes of yeasts have lowhomology with those of molds (for example, 35% in the comparison of thatof spaur1 and that of anaur1), middle regions of these amino acidsequences have high homology (57% in the comparison of the regionbetween 24th and 329th of spaur1 and the region between 59th and 364thof anaur1). Furthermore, a secondary structure predicted from amino acidsequence of these genes have identical characteristic which is membraneprotein containing transmembrane domains. Therefore, these proteins arehomologous in structure also.

50 4140 base pairs nucleic acid double linear Genomic DNA unknown 1AGATCTGTGG CTTCCGGTTG GCTACTTGTA ACCAACTGAT GGTCAGATGG ATCTGCCGTC 60TGTTTTGATT TGAATTTTCC CTGCTCATTC TGATTCTGTG AGAGGCTGCA TTCATTATCA 120CATCTCATAC CCGGCGCCTG CGACTTCGGT CACCTCTGCG GTCTGGCGGT TACCGGGGTC 180CGTCTGAGAC TCGTCAGTCA GCCATTCGAG TATGCGAACT CTGACTTTGC TCACCTAAGA 240GTTTGCACGA GATGCCGAAA TCCTCCTCGA GTAGAGTTTG CAAGGCTTGA ACCTTGGTCC 300TTGAAGCCCG AAAGTGGCTC AGTAGTGGGA TCGATAGTCT GGTTGTTGAA GATTTTCTCC 360TCCACCTTAC CTATGGCCGC TGGCCTTCTC CACCTTTCAG GCTTTCAGGC ACCCTCGGCT 420CGGATTCTGT ATCGTCCGGT ACCGAAGCTA GTCCTAGCTA GTCAAAGCTA GTCCAAGCTA 480GTCTCGTCAA GGTTTGGCGC AGCGCGGTTC CGTGTAAAGT ACAAATTTGA AATACGAATA 540CGCAGTACTC GCAGCCGGCA CTTCCGCTCA GCCCAGGCTC AGAGGCTAAG GGTGTTGGCG 600CTTCCTCATC ATCTTCTTCT CGTCGACCTT TTCCTCTTTC TCTCCCTATC GGTGCTTCTC 660TCCAACCTCA TTCTCAGTCG TTCGCCCATC AGGTTTATAC TCCGGCTCCG TGGCCATCTG 720CCTCCCTCAC GACCTCCTCG TTCCAGGTTT TCCTCTCGAC TGCTGCGCCC TTGCACTTCG 780CCTTGCATCA GTGAAACCCC CTGCAACGTG ACGGCTCAAA GACATCCTCG TTTGGCCGCT 840GGAGACCGGA GCGTGCGCTT CGTTTCGTCT TCTTCGAACC GATCTCAATT TCCCCGCTCG 900GGTTGACGCC GTCAGCACCC TGCTCGTTGC CTAACGGCTT GTTATTCAAG ACCCCTTTTC 960TGCCGCTTCC GCGACCGATT TATTCGTCGC CTTCCAACTC TTGTACAATC GGGGGGAAAG 1020AAAGCAGACG GAGTTCGATC TGGAGGAATT ATAGCTGAGT CTTGCCCGCA AGACTCGCCG 1080CAACCATGAA TCAAACACTT CCCACGTGGA AGGACCGCAC GCAGAACCAG TTTGGAAAGC 1140TTCAGATCCA GGTTCCATGG CGGTCCATCC AACTGCTCGT CCCGCATCGC ATGCGGCGGA 1200AGTTAAGGTC CAAATTGCGC AGTAGAGCGT CTCCTACCTC GTCAATAGCC TCTTTACAGA 1260CGTCGTTATC GCCTGCAGAC ACACTACGAT CGCTCCAAAG CCACCGATGG ACGGTTTACG 1320ACTTCCAATA TCTGCTTCTG TTGATCGTGG GCATCTTCTC TTTGACCGTT ATCGAGTCGC 1380CCGGGCCTTT GGGCAAAACG GCCATTTTCT CCATGCTCCT ATTCTCTCTC CTGATCCCTA 1440TGACCCGCCA GTTCTTCCTC CCGTTTCTGC CGATTGCCGG ATGGCTTCTG TTTTTCTACG 1500CCTGCCAGTG AGTTAAAAAC AACCCGCTAC CAGACCCCGT GCAGCAGTTA CTCACATATG 1560CAGGTTCATC CCAAGCGATT GGCGCCCTGC GATTTGGGTT CGTGTCTTGC CTGCACTGGA 1620GAATATTCTC TACGGCGCAA ACATCAGCAA CATCCTATCC GCTCACCAGA ACGTTGTGCT 1680TGACGTGCTG GCGTGGCTAC CCTACGGTAT CTGCCACTAT GGCGCTCCGT TTGTGTGCTC 1740GTTGATCATG TTCATCTTCG GTCCGCCCGG CACTGTTCCC CTTTTCGCGC GCACTTTCGG 1800CTATATCAGT ATGACTGCGG TTACTATTCA GCTGTTTTTC CCTTGCTCTC CACCTTGGTA 1860TGAGAATCGC TATGGTCTAG CTCCGGCAGA CTACTCCATC CAAGGTGATC CCGCAGGGCT 1920TGCCCGCATT GACAAGCTTT TCGGCATCGA CCTTTACACG TCTGTTTTCC ATCAGTCGCC 1980TGTTGTGTTC GGCGCTTTTC CGTCGCTGCA TGCTGCCGAC TCAACCCTGG CCGCACTTTT 2040CATGAGTCAT GTTTTCCCCC GCATGAAGCC CGTCTTCGTG ACCTATACTC TATGGATGTG 2100GTGGGCAACA ATGTACCTCT CACATCACTA TGCGGTCGAT TTGGTTGCGG GTGGTCTCCT 2160GGCCGCCATT GCTTTCTACT TCGCCAAGAC CCGATTCCTT CCCCGTGTCC AGCTCGACAA 2220GACCTTCCGT TGGGACTACG ACTATGTGGA ATTCGGCGAG TCTGCCCTGG AGTATGGGTA 2280TGGTGCAGCT GGCTATGATG GAGACTTCAA TCTCGACAGC GATGAATGGA CTGTTGGTTC 2340TTCATCCTCC GTCTCCTCAG GCTCCTTGAG TCCCGTTGAC GATCATTACT CATGGGAAAC 2400CGAGGCACTG ACCTCCCCAC ATACTGATAT TGAGTCCGGC AGGCATACTT TCAGCCCTTG 2460AGTAGCCACA AACCAAACTC GATACCTGCA TATAGCGATC TCGCTCCTCC TCCACTGCAT 2520CTATTTACGA GACGGCGTTA GAACATTTCA CGACATTCTG GCTTTATTGC ATCGAGCACA 2580TTTCGACACA TATATCTTTA ATACCCTTTC TTCGGTGTCC CAGATCATCG GTTCGACCTT 2640AATGTACCTC GGTCCGAATC CGCCTGGGAT ACTGTTTCTC TTTCCGCCGC ACTTCACTGT 2700ACATTGCTTG ACATTGCGAA ACCGGGTTGG GCTCGAACGT GGGATGGGTT ATCGCTCATC 2760GCTACACGCC GTTGCTCCAT CATAATGTTA ATGGACACAA TGGGGCTACG CATCCTGGTG 2820TTTAGTCCTG GAAGACCATC CGATAACCCC CGTCGGTAAC ACTCGCTTGT CTCGTGTCCA 2880CCCAGACACT ACTTCAATTC TCACTTCTAT CGTCCGCTAT TACCTTGACC TGGTCGAACC 2940CATCCTTATT ATTCGTTTCG ACTATGCTAT ATATTTATTT TTACCATTCG TGTCGATCGC 3000TCATACTCTT GGCGCTTGGG ACTGGAAGCA TTTATATTGG AAAAAATCAC GGAATGGGGC 3060GCCTTTTCTT CTTGCACTTC ACTCGCTGTG CATAGACGGT TTTACATTTC TGCTTTGCAA 3120TGCATCACGA ACTCTGCATT AGCATATAGA AAGAGGGGAA GGATGGACCT TCTTCTTGAT 3180TGCTCGCATG GTTTATCCAT TCGCTCAAAG TGGATTACGT CCACATATTA CCCGGGGGCT 3240ATACACATGG CTACTGTGTT GCTTTCTGAC ATTCGCCGGA CGTGCAAGGT TGGGAGGAGA 3300GTCTGACGCT GACGGGGCTT GTTGAAGGAT GTTCACGCGT CCCGATTTGA CCCGGCTTCG 3360ACTAACCTCA GATTCTCGAC TTGTTGGACG GTGACTTGAC TTGCTTGCTA TGGTCTGACG 3420CTCTCACACC TACCTATCAC ATCCTCCTCA CCTCACAAAT TCCGCTCATG GACACTATCC 3480TCTTCTTTTC GTTTCCCTTG GATAGTGTGT GTGTGTGTGT GGTTGGGGCA AATTATCCAT 3540AGCAGCAGTA TTATTAGTTA TAATCCGGTA GTGTTATGAT TTATGAAGGC AACTTGTATA 3600CTATTGCCAC TTTGTCCATA TCTCTTGCTT GTAATAGAAC TGACATCGCG ACGCTTCGGC 3660CACGATGCAT ATAAAAACTC TAGTCAACAC GATATTAACA AGCGAAACCA TTACGCTGTA 3720AACTATTCAG GATCGCCGCG GGCCCATCTG GGACTTGACT GTACTAAATA TGTCCTAAAG 3780CAAGCAGACT AAATATTTAA CGTGGGATAT TATTCATATA CGCATATGTA TACATAGTCA 3840TAACAAGCCA AGGGGTGGGT AGGGGTGGGT AATTATTATT TTTTTTCGTC GATACAAGTA 3900TCCATCCTTA AATGTCCGTG GTCTACTCTT CATAAATCTT AACCCGCTCC GCATACTCCT 3960TTATCCTCGA GACAAAAGTG TCTTCAATTT CATCGCCACG GCCACCAGCA ACGCGGAGGA 4020TAAGGTCGTT GAGAGAGGCG CGGCCGAGAA TGTCGACATG GTCGCCTTCA TATTGTTAGC 4080ATGCGACGTC AGACTGGAAC CAGGAAGGGA AAGGAGAGAG GTACCTGTAT TTGGACCACC 4140439 amino acids amino acid single linear peptide unknown 2 Met Asn GlnThr Leu Pro Thr Trp Lys Asp Arg Thr Gln Asn Gln 1 5 10 15 Phe Gly LysLeu Gln Ile Gln Val Pro Trp Arg Ser Ile Gln Leu 20 25 30 Leu Val Pro HisArg Met Arg Arg Lys Leu Arg Ser Lys Leu Arg 35 40 45 Ser Arg Ala Ser ProThr Ser Ser Ile Ala Ser Leu Gln Thr Ser 50 55 60 Leu Ser Pro Ala Asp ThrLeu Arg Ser Leu Gln Ser His Arg Trp 65 70 75 Thr Val Tyr Asp Phe Gln TyrLeu Leu Leu Leu Ile Val Gly Ile 80 85 90 Phe Ser Leu Thr Val Ile Glu SerPro Gly Pro Leu Gly Lys Thr 95 100 105 Ala Ile Phe Ser Met Leu Leu PheSer Leu Leu Ile Pro Met Thr 110 115 120 Arg Gln Phe Phe Leu Pro Phe LeuPro Ile Ala Gly Trp Leu Leu 125 130 135 Phe Phe Tyr Ala Cys Gln Phe IlePro Ser Asp Trp Arg Pro Ala 140 145 150 Ile Trp Val Arg Val Leu Pro AlaLeu Glu Asn Ile Leu Tyr Gly 155 160 165 Ala Asn Ile Ser Asn Ile Leu SerAla His Gln Asn Val Val Leu 170 175 180 Asp Val Leu Ala Trp Leu Pro TyrGly Ile Cys His Tyr Gly Ala 185 190 195 Pro Phe Val Cys Ser Leu Ile MetPhe Ile Phe Gly Pro Pro Gly 200 205 210 Thr Val Pro Leu Phe Ala Arg ThrPhe Gly Tyr Ile Ser Met Thr 215 220 225 Ala Val Thr Ile Gln Leu Phe PhePro Cys Ser Pro Pro Trp Tyr 230 235 240 Glu Asn Arg Tyr Gly Leu Ala ProAla Asp Tyr Ser Ile Gln Gly 245 250 255 Asp Pro Ala Gly Leu Ala Arg IleAsp Lys Leu Phe Gly Ile Asp 260 265 270 Leu Tyr Thr Ser Val Phe His GlnSer Pro Val Val Phe Gly Ala 275 280 285 Phe Pro Ser Leu His Ala Ala AspSer Thr Leu Ala Ala Leu Phe 290 295 300 Met Ser His Val Phe Pro Arg MetLys Pro Val Phe Val Thr Tyr 305 310 315 Thr Leu Trp Met Trp Trp Ala ThrMet Tyr Leu Ser His His Tyr 320 325 330 Ala Val Asp Leu Val Ala Gly GlyLeu Leu Ala Ala Ile Ala Phe 335 340 345 Tyr Phe Ala Lys Thr Arg Phe LeuPro Arg Val Gln Leu Asp Lys 350 355 360 Thr Phe Arg Trp Asp Tyr Asp TyrVal Glu Phe Gly Glu Ser Ala 365 370 375 Leu Glu Tyr Gly Tyr Gly Ala AlaGly Tyr Asp Gly Asp Phe Asn 380 385 390 Leu Asp Ser Asp Glu Trp Thr ValGly Ser Ser Ser Ser Val Ser 395 400 405 Ser Gly Ser Leu Ser Pro Val AspAsp His Tyr Ser Trp Glu Thr 410 415 420 Glu Ala Leu Thr Ser Pro His ThrAsp Ile Glu Ser Gly Arg His 425 430 435 Thr Phe Ser Pro 2856 base pairsnucleic acid double linear cDNA unknown 3 GGTTTATACT CCGGCTCCGTGGCCATCTGC CTCCCTCACG ACCTCCTCGT TCCAGGTTTT 60 CCTCTCGACT GCTGCGCCCTTGCACTTCGC CTTGCATCAG TGAAACCCCC TGCAACGTGA 120 CGGCTCAAAG ACATCCTCGTTTGGCCGCTG GAGACCGGAG CGTGCGCTTC GTTTCGTCTT 180 CTTCGAACCG ATCTCAATTTCCCCGCTCGG GTTGACGCCG TCAGCACCCT GCTCGTTGCC 240 TAACGGCTTG TTATTCAAGACCCCTTTTCT GCCGCTTCCG CGACCGATTT ATTCGTCGCC 300 TTCCAACTCT TGTACAATCGGGGGGAAAGA AAGCAGACGG AGTTCGATCT GGAGGAATTA 360 TAGCTGAGTC TTGCCCGCAAGACTCGCCGC AACCATGAAT CAAACACTTC CCACGTGGAA 420 GGACCGCACG CAGAACCAGTTTGGAAAGCT TCAGATCCAG GTTCCATGGC GGTCCATCCA 480 ACTGCTCGTC CCGCATCGCATGCGGCGGAA GTTAAGGTCC AAATTGCGCA GTAGAGCGTC 540 TCCTACCTCG TCAATAGCCTCTTTACAGAC GTCGTTATCG CCTGCAGACA CACTACGATC 600 GCTCCAAAGC CACCGATGGACGGTTTACGA CTTCCAATAT CTGCTTCTGT TGATCGTGGG 660 CATCTTCTCT TTGACCGTTATCGAGTCGCC CGGGCCTTTG GGCAAAACGG CCATTTTCTC 720 CATGCTCCTA TTCTCTCTCCTGATCCCTAT GACCCGCCAG TTCTTCCTCC CGTTTCTGCC 780 GATTGCCGGA TGGCTTCTGTTTTTCTACGC CTGCCAGTTC ATCCCAAGCG ATTGGCGCCC 840 TGCGATTTGG GTTCGTGTCTTGCCTGCACT GGAGAATATT CTCTACGGCG CAAACATCAG 900 CAACATCCTA TCCGCTCACCAGAACGTTGT GCTTGACGTG CTGGCGTGGC TACCCTACGG 960 TATCTGCCAC TATGGCGCTCCGTTTGTGTG CTCGTTGATC ATGTTCATCT TCGGTCCGCC 1020 CGGCACTGTT CCCCTTTTCGCGCGCACTTT CGGCTATATC AGTATGACTG CGGTTACTAT 1080 TCAGCTGTTT TTCCCTTGCTCTCCACCTTG GTATGAGAAT CGCTATGGTC TAGCTCCGGC 1140 AGACTACTCC ATCCAAGGTGATCCCGCAGG GCTTGCCCGC ATTGACAAGC TTTTCGGCAT 1200 CGACCTTTAC ACGTCTGTTTTCCATCAGTC GCCTGTTGTG TTCGGCGCTT TTCCGTCGCT 1260 GCATGCTGCC GACTCAACCCTGGCCGCACT TTTCATGAGT CATGTTTTCC CCCGCATGAA 1320 GCCCGTCTTC GTGACCTATACTCTATGGAT GTGGTGGGCA ACAATGTACC TCTCACATCA 1380 CTATGCGGTC GATTTGGTTGCGGGTGGTCT CCTGGCCGCC ATTGCTTTCT ACTTCGCCAA 1440 GACCCGATTC CTTCCCCGTGTCCAGCTCGA CAAGACCTTC CGTTGGGACT ACGACTATGT 1500 GGAATTCGGC GAGTCTGCCCTGGAGTATGG GTATGGTGCA GCTGGCTATG ATGGAGACTT 1560 CAATCTCGAC AGCGATGAATGGACTGTTGG TTCTTCATCC TCCGTCTCCT CAGGCTCCTT 1620 GAGTCCCGTT GACGATCATTACTCATGGGA AACCGAGGCA CTGACCTCCC CACATACTGA 1680 TATTGAGTCC GGCAGGCATACTTTCAGCCC TTGAGTAGCC ACAAACCAAA CTCGATACCT 1740 GCATATAGCG ATCTCGCTCCTCCTCCACTG CATCTATTTA CGAGACGGCG TTAGAACATT 1800 TCACGACATT CTGGCTTTATTGCATCGAGC ACATTTCGAC ACATATATCT TTAATACCCT 1860 TTCTTCGGTG TCCCAGATCATCGGTTCGAC CTTAATGTAC CTCGGTCCGA ATCCGCCTGG 1920 GATACTGTTT CTCTTTCCGCCGCACTTCAC TGTACATTGC TTGACATTGC GAAACCGGGT 1980 TGGGCTCGAA CGTGGGATGGGTTATCGCTC ATCGCTACAC GCCGTTGCTC CATCATAATG 2040 TTAATGGACA CAATGGGGCTACGCATCCTG GTGTTTAGTC CTGGAAGACC ATCCGATAAC 2100 CCCCGTCGGT AACACTCGCTTGTCTCGTGT CCACCCAGAC ACTACTTCAA TTCTCACTTC 2160 TATCGTCCGC TATTACCTTGACCTGGTCGA ACCCATCCTT ATTATTCGTT TCGACTATGC 2220 TATATATTTA TTTTTACCATTCGTGTCGAT CGCTCATACT CTTGGCGCTT GGGACTGGAA 2280 GCATTTATAT TGGAAAAAATCACGGAATGG GGCGCCTTTT CTTCTTGCAC TTCACTCGCT 2340 GTGCATAGAC GGTTTTACATTTCTGCTTTG CAATGCATCA CGAACTCTGC ATTAGCATAT 2400 AGAAAGAGGG GAAGGATGGACCTTCTTCTT GATTGCTCGC ATGGTTTATC CATTCGCTCA 2460 AAGTGGATTA CGTCCACATATTACCCGGGG GCTATACACA TGGCTACTGT GTTGCTTTCT 2520 GACATTCGCC GGACGTGCAAGGTTGGGAGG AGAGTCTGAC GCTGACGGGG CTTGTTGAAG 2580 GATGTTCACG CGTCCCGATTTGACCCGGCT TCGACTAACC TCAGATTCTC GACTTGTTGG 2640 ACGGTGACTT GACTTGCTTGCTATGGTCTG ACGCTCTCAC ACCTACCTAT CACATCCTCC 2700 TCACCTCACA AATTCCGCTCATGGACACTA TCCTCTTCTT TTCGTTTCCC TTGGATAGTG 2760 TGTGTGTGTG TGTGGTTGGGGCAAATTATC CATAGCAGCA GTATTATTAG TTATAATCCG 2820 GTAGTGTTAT GATTTATGAAGGCAACTTGT ATACTA 2856 439 amino acids amino acid single linear peptideunknown 4 Met Asn Gln Thr Leu Pro Thr Trp Lys Asp Arg Thr Gln Asn Gln 15 10 15 Phe Gly Lys Leu Gln Ile Gln Val Pro Trp Arg Ser Ile Gln Leu 2025 30 Leu Val Pro His Arg Met Arg Arg Lys Leu Arg Ser Lys Leu Arg 35 4045 Ser Arg Ala Ser Pro Thr Ser Ser Ile Ala Ser Leu Gln Thr Ser 50 55 60Leu Ser Pro Ala Asp Thr Leu Arg Ser Leu Gln Ser His Arg Trp 65 70 75 ThrVal Tyr Asp Phe Gln Tyr Leu Leu Leu Leu Ile Val Gly Ile 80 85 90 Phe SerLeu Thr Val Ile Glu Ser Pro Gly Pro Leu Gly Lys Thr 95 100 105 Ala IlePhe Ser Met Leu Leu Phe Ser Leu Leu Ile Pro Met Thr 110 115 120 Arg GlnPhe Phe Leu Pro Phe Leu Pro Ile Ala Gly Trp Leu Leu 125 130 135 Phe PheTyr Ala Cys Gln Phe Ile Pro Ser Asp Trp Arg Pro Ala 140 145 150 Ile TrpVal Arg Val Leu Pro Ala Leu Glu Asn Ile Leu Tyr Gly 155 160 165 Ala AsnIle Ser Asn Ile Leu Ser Ala His Gln Asn Val Val Leu 170 175 180 Asp ValLeu Ala Trp Leu Pro Tyr Gly Ile Cys His Tyr Gly Ala 185 190 195 Pro PheVal Cys Ser Leu Ile Met Phe Ile Phe Gly Pro Pro Gly 200 205 210 Thr ValPro Leu Phe Ala Arg Thr Phe Gly Tyr Ile Ser Met Thr 215 220 225 Ala ValThr Ile Gln Leu Phe Phe Pro Cys Ser Pro Pro Trp Tyr 230 235 240 Glu AsnArg Tyr Gly Leu Ala Pro Ala Asp Tyr Ser Ile Gln Gly 245 250 255 Asp ProAla Gly Leu Ala Arg Ile Asp Lys Leu Phe Gly Ile Asp 260 265 270 Leu TyrThr Ser Gly Phe His Gln Ser Pro Val Val Phe Gly Ala 275 280 285 Phe ProSer Leu His Ala Ala Asp Ser Thr Leu Ala Ala Leu Phe 290 295 300 Met SerHis Val Phe Pro Arg Met Lys Pro Val Phe Val Thr Tyr 305 310 315 Thr LeuTrp Met Trp Trp Ala Thr Met Tyr Leu Ser His His Tyr 320 325 330 Ala ValAsp Leu Val Ala Gly Gly Leu Leu Ala Ala Ile Ala Phe 335 340 345 Tyr PheAla Lys Thr Arg Phe Leu Pro Arg Val Gln Leu Asp Lys 350 355 360 Thr PheArg Trp Asp Tyr Asp Tyr Val Glu Phe Gly Glu Ser Ala 365 370 375 Leu GluTyr Gly Tyr Gly Ala Ala Gly Tyr Asp Gly Asp Phe Asn 380 385 390 Leu AspSer Asp Glu Trp Thr Val Gly Ser Ser Ser Ser Val Ser 395 400 405 Ser GlySer Leu Ser Pro Val Asp Asp His Tyr Ser Trp Glu Thr 410 415 420 Glu AlaLeu Thr Ser Pro His Thr Asp Ile Glu Ser Gly Arg His 425 430 435 Thr PheSer Pro 436 amino acids amino acid single linear peptide unknown 5 MetAsn Thr Thr Leu Pro Ser Trp Lys Asp Arg Thr Gln Asn Gln 1 5 10 15 PheGly Lys Leu Gln Ile Gln Val Pro Trp Arg Thr Ile Gln Leu 20 25 30 Leu ValPro His Arg Met Arg Arg Lys Ile Arg Ser Lys Leu Arg 35 40 45 Ser Arg IleSer Pro Thr Ser Ser Ile Ser Ser Leu Gln Thr Ser 50 55 60 Phe Ser Pro ValAsp Thr Leu Arg Ser Leu Gln Ser His Arg Trp 65 70 75 Thr Leu Tyr Asp PheGln Tyr Leu Leu Leu Leu Ile Val Gly Ile 80 85 90 Phe Ser Leu Ser Val MetGlu Ser Pro Gly Pro Leu Ala Lys Thr 95 100 105 Ala Ala Phe Thr Leu LeuLeu Val Ser Leu Leu Leu Pro Ile Thr 110 115 120 Arg Gln Phe Phe Leu ProPhe Leu Pro Ile Ala Gly Trp Leu Ile 125 130 135 Phe Phe Tyr Ala Cys GlnPhe Ile Pro Ser Asp Trp Arg Pro Ala 140 145 150 Ile Trp Val Arg Val LeuPro Ala Leu Glu Asn Ile Leu Tyr Gly 155 160 165 Ala Asn Ile Ser Asn IleLeu Ser Ala His Gln Asn Val Val Leu 170 175 180 Asp Val Leu Ala Trp LeuPro Tyr Gly Ile Cys His Tyr Gly Ala 185 190 195 Pro Phe Val Cys Ser AlaIle Met Phe Ile Phe Gly Pro Pro Gly 200 205 210 Thr Val Pro Leu Phe AlaArg Thr Phe Gly Tyr Ile Ser Met Ala 215 220 225 Ala Val Thr Ile Gln LeuPhe Phe Pro Cys Ser Pro Pro Trp Tyr 230 235 240 Glu Asn Leu Tyr Gly LeuAla Pro Ala Asp Tyr Ser Met Pro Gly 245 250 255 Asn Pro Ala Gly Leu AlaArg Ile Asp Glu Leu Phe Gly Ile Asp 260 265 270 Leu Tyr Thr Ser Gly PheArg Gln Ser Pro Val Val Phe Gly Ala 275 280 285 Phe Pro Ser Leu His AlaAla Asp Ser Thr Leu Ala Ala Leu Phe 290 295 300 Met Ser Gln Val Phe ProArg Leu Lys Pro Leu Phe Val Ile Tyr 305 310 315 Thr Leu Trp Met Trp TrpAla Thr Met Tyr Leu Ser His His Tyr 320 325 330 Ala Val Asp Leu Val GlyGly Gly Leu Leu Ala Thr Val Ala Phe 335 340 345 Tyr Phe Ala Lys Thr ArgPhe Met Pro Arg Val Gln Asn Asp Lys 350 355 360 Met Phe Arg Trp Asp TyrAsp Tyr Val Glu Tyr Gly Asp Ser Ala 365 370 375 Leu Asp Tyr Gly Tyr GlyPro Ala Ser Phe Glu Gly Glu Phe Asn 380 385 390 Leu Asp Ser Asp Glu TrpThr Val Gly Ser Ser Ser Ser Ile Ser 395 400 405 Ser Gly Ser Leu Ser ProVal Asp Asp His Tyr Ser Trp Glu Gly 410 415 420 Glu Thr Leu Ala Ser ProAla Thr Asp Ile Glu Ser Gly Arg His 425 430 435 Phe 401 amino acidsamino acid single linear peptide unknown 6 Met Ala Asn Pro Phe Ser ArgTrp Phe Leu Ser Glu Arg Pro Pro 1 5 10 15 Asn Cys His Val Ala Asp LeuGlu Thr Ser Leu Asp Pro His Gln 20 25 30 Thr Leu Leu Lys Val Gln Lys TyrLys Pro Ala Leu Ser Asp Trp 35 40 45 Val His Tyr Ile Phe Leu Gly Ser IleMet Leu Phe Val Phe Ile 50 55 60 Thr Asn Pro Ala Pro Trp Ile Phe Lys IleLeu Phe Tyr Cys Phe 65 70 75 Leu Gly Thr Leu Phe Ile Ile Pro Ala Thr SerGln Phe Phe Phe 80 85 90 Asn Ala Leu Pro Ile Leu Thr Trp Val Ala Leu TyrPhe Thr Ser 95 100 105 Ser Tyr Phe Pro Asp Asp Arg Arg Pro Pro Ile ThrVal Lys Val 110 115 120 Leu Pro Ala Val Glu Thr Ile Leu Tyr Gly Asp AsnLeu Ser Asp 125 130 135 Ile Leu Ala Thr Ser Thr Asn Ser Phe Leu Asp IleLeu Ala Trp 140 145 150 Leu Pro Tyr Gly Leu Phe His Phe Gly Ala Pro PheVal Val Ala 155 160 165 Ala Ile Leu Phe Val Phe Gly Pro Pro Thr Val LeuGln Gly Tyr 170 175 180 Ala Phe Ala Phe Gly Tyr Met Asn Leu Phe Gly ValIle Met Gln 185 190 195 Asn Val Phe Pro Ala Ala Pro Pro Trp Tyr Lys IleLeu Tyr Gly 200 205 210 Leu Gln Ser Ala Asn Tyr Asp Met His Gly Ser ProGly Gly Leu 215 220 225 Ala Arg Ile Asp Lys Leu Leu Gly Ile Asn Met TyrThr Thr Ala 230 235 240 Phe Ser Asn Ser Ser Val Ile Phe Gly Ala Phe ProSer Leu His 245 250 255 Ser Gly Cys Ala Thr Met Glu Ala Leu Phe Phe CysTyr Cys Phe 260 265 270 Pro Lys Leu Lys Pro Leu Phe Ile Ala Tyr Val CysTrp Leu Trp 275 280 285 Trp Ser Thr Met Tyr Leu Thr His His Tyr Phe ValAsp Leu Met 290 295 300 Ala Gly Ser Val Leu Ser Tyr Val Ile Phe Gln TyrThr Lys Tyr 305 310 315 Thr His Leu Pro Ile Val Asp Thr Ser Leu Phe CysArg Trp Ser 320 325 330 Tyr Thr Ser Ile Glu Lys Tyr Asp Ile Ser Lys SerAsp Pro Leu 335 340 345 Ala Ala Asp Ser Asn Asp Ile Glu Ser Val Pro LeuSer Asn Leu 350 355 360 Glu Leu Asp Phe Asp Leu Asn Met Thr Asp Glu ProSer Val Ser 365 370 375 Pro Ser Leu Phe Asp Gly Ser Thr Ser Val Ser ArgSer Ser Ala 380 385 390 Thr Ser Ile Thr Ser Leu Gly Val Lys Arg Ala 395400 422 amino acids amino acid single linear peptide unknown 7 Met SerAla Leu Ser Thr Leu Lys Lys Arg Leu Ala Ala Cys Asn 1 5 10 15 Arg AlaSer Gln Tyr Lys Leu Glu Thr Ser Leu Asn Pro Met Pro 20 25 30 Thr Phe ArgLeu Leu Arg Asn Thr Lys Trp Ser Trp Thr His Leu 35 40 45 Gln Tyr Val PheLeu Ala Gly Asn Leu Ile Phe Ala Cys Ile Val 50 55 60 Ile Glu Ser Pro GlyPhe Trp Gly Lys Phe Gly Ile Ala Cys Leu 65 70 75 Leu Ala Ile Ala Leu ThrVal Pro Leu Thr Arg Gln Ile Phe Phe 80 85 90 Pro Ala Ile Val Ile Ile ThrTrp Ala Ile Leu Phe Tyr Ser Cys 95 100 105 Arg Phe Ile Pro Glu Arg TrpArg Pro Pro Ile Trp Val Arg Val 110 115 120 Leu Pro Thr Leu Glu Asn IleLeu Tyr Gly Ser Asn Leu Ser Ser 125 130 135 Leu Leu Ser Lys Thr Thr HisSer Ile Leu Asp Ile Leu Ala Trp 140 145 150 Val Pro Tyr Gly Val Met HisTyr Ser Ala Pro Phe Ile Ile Ser 155 160 165 Phe Ile Leu Phe Ile Phe AlaPro Pro Gly Thr Leu Pro Val Trp 170 175 180 Ala Arg Thr Phe Gly Tyr MetAsn Leu Phe Gly Val Leu Ile Gln 185 190 195 Met Ala Phe Pro Cys Ser ProPro Trp Tyr Glu Asn Met Tyr Gly 200 205 210 Leu Glu Pro Ala Thr Tyr AlaVal Arg Gly Ser Pro Gly Gly Leu 215 220 225 Ala Arg Ile Asp Ala Leu PheGly Thr Ser Ile Tyr Thr Asp Gly 230 235 240 Phe Ser Asn Ser Pro Val ValPhe Gly Ala Phe Pro Ser Leu His 245 250 255 Ala Gly Trp Ala Met Leu GluAla Leu Phe Leu Ser His Val Phe 260 265 270 Pro Arg Tyr Arg Phe Cys PheTyr Gly Tyr Val Leu Trp Leu Cys 275 280 285 Trp Cys Thr Met Tyr Leu ThrHis His Tyr Phe Val Asp Leu Val 290 295 300 Gly Gly Met Cys Leu Ala IleIle Cys Phe Val Phe Ala Gln Lys 305 310 315 Leu Arg Leu Pro Gln Leu GlnThr Gly Lys Ile Leu Arg Trp Glu 320 325 330 Tyr Glu Phe Val Ile His GlyHis Gly Leu Ser Glu Lys Thr Ser 335 340 345 Asn Ser Leu Ala Arg Thr GlySer Pro Tyr Leu Leu Gly Arg Asp 350 355 360 Ser Phe Thr Gln Asn Pro AsnAla Val Ala Phe Met Ser Gly Leu 365 370 375 Asn Asn Met Glu Leu Ala AsnThr Asp His Glu Trp Ser Val Gly 380 385 390 Ser Ser Ser Pro Glu Pro LeuPro Ser Pro Ala Ala Asp Leu Ile 395 400 405 Asp Arg Pro Ala Ser Thr ThrSer Ser Ile Phe Asp Ala Ser His 410 415 420 Leu Pro 471 amino acidsamino acid single linear peptide unknown 8 Met Ala Ser Ser Ile Leu ArgSer Lys Ile Ile Gln Lys Pro Tyr 1 5 10 15 Gln Leu Phe His Tyr Tyr PheLeu Ser Glu Lys Ala Pro Gly Ser 20 25 30 Thr Val Ser Asp Leu Asn Phe AspThr Asn Ile Gln Thr Ser Leu 35 40 45 Arg Lys Leu Lys His His His Trp ThrVal Gly Glu Ile Phe His 50 55 60 Tyr Gly Phe Leu Val Ser Ile Leu Phe PheVal Phe Val Val Phe 65 70 75 Pro Ala Ser Phe Phe Ile Lys Leu Pro Ile IleLeu Ala Phe Ala 80 85 90 Thr Cys Phe Leu Ile Pro Leu Thr Ser Gln Phe PheLeu Pro Ala 95 100 105 Leu Pro Val Phe Thr Trp Leu Ala Leu Tyr Phe ThrCys Ala Lys 110 115 120 Ile Pro Gln Glu Trp Lys Pro Ala Ile Thr Val LysVal Leu Pro 125 130 135 Ala Met Glu Thr Ile Leu Tyr Gly Asp Asn Leu SerAsn Val Leu 140 145 150 Ala Thr Ile Thr Thr Gly Val Leu Asp Ile Leu AlaTrp Leu Pro 155 160 165 Tyr Gly Ile Ile His Phe Ser Phe Pro Phe Val LeuAla Ala Ile 170 175 180 Ile Phe Leu Phe Gly Pro Pro Thr Ala Leu Arg SerPhe Gly Phe 185 190 195 Ala Phe Gly Tyr Met Asn Leu Leu Gly Val Leu IleGln Met Ala 200 205 210 Phe Pro Ala Ala Pro Pro Trp Tyr Lys Asn Leu HisGly Leu Glu 215 220 225 Pro Ala Asn Tyr Ser Met His Gly Ser Pro Gly GlyLeu Gly Arg 230 235 240 Ile Asp Lys Leu Leu Gly Val Asp Met Tyr Thr ThrGly Phe Ser 245 250 255 Asn Ser Ser Ile Ile Phe Gly Ala Phe Pro Ser LeuHis Ser Gly 260 265 270 Cys Cys Ile Met Glu Val Leu Phe Leu Cys Trp LeuPhe Pro Arg 275 280 285 Phe Lys Phe Val Trp Val Thr Tyr Ala Ser Trp LeuTrp Trp Ser 290 295 300 Thr Met Tyr Leu Thr His His Tyr Phe Val Asp LeuIle Gly Gly 305 310 315 Ala Met Leu Ser Leu Thr Val Phe Glu Phe Thr LysTyr Lys Tyr 320 325 330 Leu Pro Lys Asn Lys Glu Gly Leu Phe Cys Arg TrpSer Tyr Thr 335 340 345 Glu Ile Glu Lys Ile Asp Ile Gln Glu Ile Asp ProLeu Ser Tyr 350 355 360 Asn Tyr Ile Pro Val Asn Ser Asn Asp Asn Glu SerArg Leu Tyr 365 370 375 Thr Arg Val Tyr Gln Glu Ser Gln Val Ser Pro ProGln Arg Ala 380 385 390 Glu Thr Pro Glu Ala Phe Glu Met Ser Asn Phe SerArg Ser Arg 395 400 405 Gln Ser Ser Lys Thr Gln Val Pro Leu Ser Asn LeuThr Asn Asn 410 415 420 Asp Gln Val Ser Gly Ile Asn Glu Glu Asp Glu GluGlu Glu Gly 425 430 435 Asp Glu Ile Ser Ser Ser Thr Pro Ser Val Phe GluAsp Glu Pro 440 445 450 Gln Gly Ser Thr Tyr Ala Ala Ser Ser Ala Thr SerVal Asp Asp 455 460 465 Leu Asp Ser Lys Arg Asn 470 10 amino acids aminoacid single linear peptide unknown /note= “Xaa at position 3 is Val orIle” /note= “Xaa at position 7 is Leu or Val” 9 Leu Asp Xaa Leu Ala TrpXaa Pro Tyr Gly 1 5 10 8 amino acids amino acid single linear peptideunknown 10 Phe Gly Ala Phe Pro Ser Leu His 1 5 12 amino acids amino acidsingle linear peptide unknown /note= “Xaa at position 5 is Ser or Thr”/note= “Xaa at position 9 is Ala or Phe” 11 Thr Met Tyr Leu Xaa His HisTyr Xaa Val Asp Leu 1 5 10 2935 base pairs nucleic acid double linearcDNA unknown 12 ATTTTCTTCC CCATAACAAC TCTTCTCGCC CTTCCTCCGG CTCCGTGGCCAAATTGTTTT 60 ATGCAGCGCC TCCTAGCGAT TTAACCTCGT TCTCGTTGCC CTTGCCTGTCCGCCTTGCGT 120 CAGTACGACC CTTGCAACGT GACCTTCCCC AGAGTATCCT CGTTTGGCCGCTGGAGACCG 180 GAGCTTGCAC CCTCATAAAC TAGCTCTTCG AAATCAATTC TCCGTTCTCCAGAGATTATC 240 GGATCGAATC TCTCCGCTGT CGACACCTTT CGTCTCTCGG TGATCCTCGCCCTTGGAGTC 300 TCGTCACGTT GACGCCTTGA ACCCCTGGCC GCCAACTCCA CATAGGAGACCACACTTCAT 360 TCTTCCCCCG CCATAATTGC AGCACCCTCC GTCTCCCTTC GAGCTCCTCCTGGATCATCA 420 AGTCCGAAAG GATTAGACTC GTCGCAGCGA TGAATACCAC CCTTCCATCCTGGAAGGATC 480 GGACGCAAAA CCAGTTCGGC AAGCTCCAGA TCCAAGTCCC ATGGCGCACCATACAACTTC 540 TCGTGCCGCA CCGTATGCGA CGGAAGATTC GGTCCAAGCT GCGCAGTCGGATCTCGCCTA 600 CCTCATCGAT ATCCTCGTTG CAGACGTCAT TCTCACCTGT CGATACACTCAGGTCGCTGC 660 AAAGTCATAG ATGGACGCTC TATGACTTTC AGTATCTTTT GCTGCTGATTGTCGGCATAT 720 TCTCGCTGAG CGTTATGGAA TCACCTGGAC CATTGGCAAA GACCGCCGCGTTTACGCTAC 780 TTCTCGTCTC TCTCCTTCTC CCGATTACGC GCCAGTTCTT CTTGCCATTCCTCCCGATTG 840 CAGGATGGCT TATATTTTTC TACGCTTGCC AGTTCATCCC GAGCGACTGGCGCCCTGCAA 900 TCTGGGTTCG CGTGCTGCCG GCTCTGGAAA ACATTCTCTA CGGTGCTAATATCAGTAACA 960 TCCTTTCCGC TCACCAAAAT GTGGTGCTTG ACGTTTTGGC GTGGCTTCCCTACGGAATCT 1020 GCCATTATGG CGCGCCATTT GTGTGCTCAG CGATCATGTT CATCTTTGGTCCTCCCGGCA 1080 CCGTCCCCCT TTTCGCTCGA ACTTTTGGAT ACATCAGCAT GGCTGCAGTCACCATTCAGC 1140 TGTTTTTCCC CTGCTCTCCT CCGTGGTACG AAAATCTGTA TGGTTTGGCTCCGGCTGATT 1200 ACTCCATGCC GGGTAATCCT GCGGGCCTTG CTCGCATCGA TGAGCTTTTTGGGATAGACT 1260 TGTACACATC GGGCTTCAGA CAATCTCCCG TCGTGTTTGG CGCATTTCCTTCCCTACATG 1320 CCGCTGATTC GACACTTGCA GCTCTATTTA TGAGCCAAGT GTTCCCACGGTTGAAGCCCT 1380 TGTTTGTCAT CTATACTCTC TGGATGTGGT GGGCTACAAT GTATCTTTCGCACCACTACG 1440 CTGTTGATCT GGTCGGTGGT GGCCTCTTGG CAACTGTCGC GTTCTACTTTGCTAAAACGC 1500 GGTTCATGCC TCGCGTCCAG AATGATAAGA TGTTCCGCTG GGACTACGATTATGTTGAGT 1560 ACGGCGATTC CGCACTCGAC TATGGGTACG GTCCAGCCAG CTTCGAAGGCGAATTCAACC 1620 TTGATAGCGA TGAGTGGACC GTTGGTTCTT CGTCATCCAT TTCGTCCGGCTCCCTCAGTC 1680 CAGTTGACGA CCACTACTCT TGGGAGGGCG AGACTCTTGC CTCTCCTGCCACCGACATCG 1740 AGTCTGGAAG GCATTTCTGA TCCTGCTCAA TGAGCCTTGA TACGTACTACACTGTGTACG 1800 TGCTACTGCA TTGACTAATG AGACGGCGTT TTCAAACAAA TTTTAACGACATGCTTGGTT 1860 ATCGCATTGA GCTGATTTCG ACACATATAT ATGTTTAATA CGTTTTGGGGACACTCCAGG 1920 GATTCATGAC GGTTGCTTCA TATCCCGACC TGGGGATGGA TTGACCTGGTTGTGCCCAAT 1980 TTTCTTCTGC CTAACGTTTT GATTATACAT GCATTTTTCA CGAAACCAGCCGGCCCGCCA 2040 TGATCGTGAC CTCAATTTGA GCTCGAATCT TCCTGGGGCC TCCAGCGATAATTCTTAATG 2100 CTCGTTCCGA GGGTGCCACA TCGGACATTC GCTTGTACAA CTTTTGCAGAACGAACATTT 2160 TCACCGATTC CAACTTGAGT CATTCGCTTA CTACTTTCAA CTGGTCGAGAAACTTCGCTT 2220 CTTTTCAGCT CGGCTAGGTG CATAAATATT TTACATTCGT GTCGATCGCTCACATTTCAC 2280 GGCGCCTGGA AACTTGGGGG TTTCGATTTC ATTGGAAAGG ATAAACAACATGGGCTGGGC 2340 GCCTTTTACA CGCACTACAT TCGCTTAGAA AAGTTCTGAT GCTTTTAATGATTCTTGCAT 2400 TGGCATATAG AAAGGGGTCC TCCAGACTCG CTACTGTGGT CCTCTCTCAACCCCACACTC 2460 GCTTGCTTTT AACAGTGGAC ACCCCGTGGA GCTACGTCTC CATCAAATATTTGGCATCAA 2520 CCGGAATCGA TGCCAGGAGG ACTGAGCTTA CTCACGGTGA CCGTCGGGTAAAAGGCGTTC 2580 ATACAGAACA TCTCCTCTAT CCTCCTGTCC CATCTCGATT CTCTGGCTGCTGGACGCAAC 2640 ACACCTCGCT GCACGTTTTC GACTTCCTAA TACGACCTAA TATCATTCTCGGTTTTCTTT 2700 GCTCTGGCTC GCGGCCGCCA TTTATATGGC GTGTCGGCTC GAGTCTGAAGTGAACTTTTT 2760 TCTCCTTTCT GGCCTCCACA ACTTTCCGAT CCCTAGCAGC TTCCCGTGCACAGCGAGGTG 2820 TTGTTGGATG ATTGTTCCAT AGCATTATCA TTATTCCTAA TCCGGTAGCGTTATGATTTA 2880 TGAAGAACAG TGATGTACAT TATTATGCGG TGATCAAAAA AAAAAAAAAAAAAAA 2935 2856 base pairs nucleic acid double linear Genomic DNA Yesunknown 13 TAGTATACAA GTTGCCTTCA TAAATCATAA CACTACCGGA TTATAACTAATAATACTGCT 60 GCTATGGATA ATTTGCCCCA ACCACACACA CACACACACT ATCCAAGGGAAACGAAAAGA 120 AGAGGATAGT GTCCATGAGC GGAATTTGTG AGGTGAGGAG GATGTGATAGGTAGGTGTGA 180 GAGCGTCAGA CCATAGCAAG CAAGTCAAGT CACCGTCCAA CAAGTCGAGAATCTGAGGTT 240 AGTCGAAGCC GGGTCAAATC GGGACGCGTG AACATCCTTC AACAAGCCCCGTCAGCGTCA 300 GACTCTCCTC CCAACCTTGC ACGTCCGGCG AATGTCAGAA AGCAACACAGTAGCCATGTG 360 TATAGCCCCC GGGTAATATG TGGACGTAAT CCACTTTGAG CGAATGGATAAACCATGCGA 420 GCAATCAAGA AGAAGGTCCA TCCTTCCCCT CTTTCTATAT GCTAATGCAGAGTTCGTGAT 480 GCATTGCAAA GCAGAAATGT AAAACCGTCT ATGCACAGCG AGTGAAGTGCAAGAAGAAAA 540 GGCGCCCCAT TCCGTGATTT TTTCCAATAT AAATGCTTCC AGTCCCAAGCGCCAAGAGTA 600 TGAGCGATCG ACACGAATGG TAAAAATAAA TATATAGCAT AGTCGAAACGAATAATAAGG 660 ATGGGTTCGA CCAGGTCAAG GTAATAGCGG ACGATAGAAG TGAGAATTGAAGTAGTGTCT 720 GGGTGGACAC GAGACAAGCG AGTGTTACCG ACGGGGGTTA TCGGATGGTCTTCCAGGACT 780 AAACACCAGG ATGCGTAGCC CCATTGTGTC CATTAACATT ATGATGGAGCAACGGCGTGT 840 AGCGATGAGC GATAACCCAT CCCACGTTCG AGCCCAACCC GGTTTCGCAATGTCAAGCAA 900 TGTACAGTGA AGTGCGGCGG AAAGAGAAAC AGTATCCCAG GCGGATTCGGACCGAGGTAC 960 ATTAAGGTCG AACCGATGAT CTGGGACACC GAAGAAAGGG TATTAAAGATATATGTGTCG 1020 AAATGTGCTC GATGCAATAA AGCCAGAATG TCGTGAAATG TTCTAACGCCGTCTCGTAAA 1080 TAGATGCAGT GGAGGAGGAG CGAGATCGCT ATATGCAGGT ATCGAGTTTGGTTTGTGGCT 1140 ACTCAAGGGC TGAAAGTATG CCTGCCGGAC TCAATATCAG TATGTGGGGAGGTCAGTGCC 1200 TCGGTTTCCC ATGAGTAATG ATCGTCAACG GGACTCAAGG AGCCTGAGGAGACGGAGGAT 1260 GAAGAACCAA CAGTCCATTC ATCGCTGTCG AGATTGAAGT CTCCATCATAGCCAGCTGCA 1320 CCATACCCAT ACTCCAGGGC AGACTCGCCG AATTCCACAT AGTCGTAGTCCCAACGGAAG 1380 GTCTTGTCGA GCTGGACACG GGGAAGGAAT CGGGTCTTGG CGAAGTAGAAAGCAATGGCG 1440 GCCAGGAGAC CACCCGCAAC CAAATCGACC GCATAGTGAT GTGAGAGGTACATTGTTGCC 1500 CACCACATCC ATAGAGTATA GGTCACGAAG ACGGGCTTCA TGCGGGGGAAAACATGACTC 1560 ATGAAAAGTG CGGCCAGGGT TGAGTCGGCA GCATGCAGCG ACGGAAAAGCGCCGAACACA 1620 ACAGGCGACT GATGGAAAAC AGACGTGTAA AGGTCGATGC CGAAAAGCTTGTCAATGCGG 1680 GCAAGCCCTG CGGGATCACC TTGGATGGAG TAGTCTGCCG GAGCTAGACCATAGCGATTC 1740 TCATACCAAG GTGGAGAGCA AGGGAAAAAC AGCTGAATAG TAACCGCAGTCATACTGATA 1800 TAGCCGAAAG TGCGCGCGAA AAGGGGAACA GTGCCGGGCG GACCGAAGATGAACATGATC 1860 AACGAGCACA CAAACGGAGC GCCATAGTGG CAGATACCGT AGGGTAGCCACGCCAGCACG 1920 TCAAGCACAA CGTTCTGGTG AGCGGATAGG ATGTTGCTGA TGTTTGCGCCGTAGAGAATA 1980 TTCTCCAGTG CAGGCAAGAC ACGAACCCAA ATCGCAGGGC GCCAATCGCTTGGGATGAAC 2040 TGGCAGGCGT AGAAAAACAG AAGCCATCCG GCAATCGGCA GAAACGGGAGGAAGAACTGG 2100 CGGGTCATAG GGATCAGGAG AGAGAATAGG AGCATGGAGA AAATGGCCGTTTTGCCCAAA 2160 GGCCCGGGCG ACTCGATAAC GGTCAAAGAG AAGATGCCCA CGATCAACAGAAGCAGATAT 2220 TGGAAGTCGT AAACCGTCCA TCGGTGGCTT TGGAGCGATC GTAGTGTGTCTGCAGGCGAT 2280 AACGACGTCT GTAAAGAGGC TATTGACGAG GTAGGAGACG CTCTACTGCGCAATTTGGAC 2340 CTTAACTTCC GCCGCATGCG ATGCGGGACG AGCAGTTGGA TGGACCGCCATGGAACCTGG 2400 ATCTGAAGCT TTCCAAACTG GTTCTGCGTG CGGTCCTTCC ACGTGGGAAGTGTTTGATTC 2460 ATGGTTGCGG CGAGTCTTGC GGGCAAGACT CAGCTATAAT TCCTCCAGATCGAACTCCGT 2520 CTGCTTTCTT TCCCCCCGAT TGTACAAGAG TTGGAAGGCG ACGAATAAATCGGTCGCGGA 2580 AGCGGCAGAA AAGGGGTCTT GAATAACAAG CCGTTAGGCA ACGAGCAGGGTGCTGACGGC 2640 GTCAACCCGA GCGGGGAAAT TGAGATCGGT TCGAAGAAGA CGAAACGAAGCGCACGCTCC 2700 GGTCTCCAGC GGCCAAACGA GGATGTCTTT GAGCCGTCAC GTTGCAGGGGGTTTCACTGA 2760 TGCAAGGCGA AGTGCAAGGG CGCAGCAGTC GAGAGGAAAA CCTGGAACGAGGAGGTCGTG 2820 AGGGAGGCAG ATGGCCACGG AGCCGGAGTA TAAACC 2856 2856 basesnucleic acid single linear mRNA Yes unknown 14 UAGUAUACAA GUUGCCUUCAUAAAUCAUAA CACUACCGGA UUAUAACUAA UAAUACUGCU 60 GCUAUGGAUA AUUUGCCCCAACCACACACA CACACACACU AUCCAAGGGA AACGAAAAGA 120 AGAGGAUAGU GUCCAUGAGCGGAAUUUGUG AGGUGAGGAG GAUGUGAUAG GUAGGUGUGA 180 GAGCGUCAGA CCAUAGCAAGCAAGUCAAGU CACCGUCCAA CAAGUCGAGA AUCUGAGGUU 240 AGUCGAAGCC GGGUCAAAUCGGGACGCGUG AACAUCCUUC AACAAGCCCC GUCAGCGUCA 300 GACUCUCCUC CCAACCUUGCACGUCCGGCG AAUGUCAGAA AGCAACACAG UAGCCAUGUG 360 UAUAGCCCCC GGGUAAUAUGUGGACGUAAU CCACUUUGAG CGAAUGGAUA AACCAUGCGA 420 GCAAUCAAGA AGAAGGUCCAUCCUUCCCCU CUUUCUAUAU GCUAAUGCAG AGUUCGUGAU 480 GCAUUGCAAA GCAGAAAUGUAAAACCGUCU AUGCACAGCG AGUGAAGUGC AAGAAGAAAA 540 GGCGCCCCAU UCCGUGAUUUUUUCCAAUAU AAAUGCUUCC AGUCCCAAGC GCCAAGAGUA 600 UGAGCGAUCG ACACGAAUGGUAAAAAUAAA UAUAUAGCAU AGUCGAAACG AAUAAUAAGG 660 AUGGGUUCGA CCAGGUCAAGGUAAUAGCGG ACGAUAGAAG UGAGAAUUGA AGUAGUGUCU 720 GGGUGGACAC GAGACAAGCGAGUGUUACCG ACGGGGGUUA UCGGAUGGUC UUCCAGGACU 780 AAACACCAGG AUGCGUAGCCCCAUUGUGUC CAUUAACAUU AUGAUGGAGC AACGGCGUGU 840 AGCGAUGAGC GAUAACCCAUCCCACGUUCG AGCCCAACCC GGUUUCGCAA UGUCAAGCAA 900 UGUACAGUGA AGUGCGGCGGAAAGAGAAAC AGUAUCCCAG GCGGAUUCGG ACCGAGGUAC 960 AUUAAGGUCG AACCGAUGAUCUGGGACACC GAAGAAAGGG UAUUAAAGAU AUAUGUGUCG 1020 AAAUGUGCUC GAUGCAAUAAAGCCAGAAUG UCGUGAAAUG UUCUAACGCC GUCUCGUAAA 1080 UAGAUGCAGU GGAGGAGGAGCGAGAUCGCU AUAUGCAGGU AUCGAGUUUG GUUUGUGGCU 1140 ACUCAAGGGC UGAAAGUAUGCCUGCCGGAC UCAAUAUCAG UAUGUGGGGA GGUCAGUGCC 1200 UCGGUUUCCC AUGAGUAAUGAUCGUCAACG GGACUCAAGG AGCCUGAGGA GACGGAGGAU 1260 GAAGAACCAA CAGUCCAUUCAUCGCUGUCG AGAUUGAAGU CUCCAUCAUA GCCAGCUGCA 1320 CCAUACCCAU ACUCCAGGGCAGACUCGCCG AAUUCCACAU AGUCGUAGUC CCAACGGAAG 1380 GUCUUGUCGA GCUGGACACGGGGAAGGAAU CGGGUCUUGG CGAAGUAGAA AGCAAUGGCG 1440 GCCAGGAGAC CACCCGCAACCAAAUCGACC GCAUAGUGAU GUGAGAGGUA CAUUGUUGCC 1500 CACCACAUCC AUAGAGUAUAGGUCACGAAG ACGGGCUUCA UGCGGGGGAA AACAUGACUC 1560 AUGAAAAGUG CGGCCAGGGUUGAGUCGGCA GCAUGCAGCG ACGGAAAAGC GCCGAACACA 1620 ACAGGCGACU GAUGGAAAACAGACGUGUAA AGGUCGAUGC CGAAAAGCUU GUCAAUGCGG 1680 GCAAGCCCUG CGGGAUCACCUUGGAUGGAG UAGUCUGCCG GAGCUAGACC AUAGCGAUUC 1740 UCAUACCAAG GUGGAGAGCAAGGGAAAAAC AGCUGAAUAG UAACCGCAGU CAUACUGAUA 1800 UAGCCGAAAG UGCGCGCGAAAAGGGGAACA GUGCCGGGCG GACCGAAGAU GAACAUGAUC 1860 AACGAGCACA CAAACGGAGCGCCAUAGUGG CAGAUACCGU AGGGUAGCCA CGCCAGCACG 1920 UCAAGCACAA CGUUCUGGUGAGCGGAUAGG AUGUUGCUGA UGUUUGCGCC GUAGAGAAUA 1980 UUCUCCAGUG CAGGCAAGACACGAACCCAA AUCGCAGGGC GCCAAUCGCU UGGGAUGAAC 2040 UGGCAGGCGU AGAAAAACAGAAGCCAUCCG GCAAUCGGCA GAAACGGGAG GAAGAACUGG 2100 CGGGUCAUAG GGAUCAGGAGAGAGAAUAGG AGCAUGGAGA AAAUGGCCGU UUUGCCCAAA 2160 GGCCCGGGCG ACUCGAUAACGGUCAAAGAG AAGAUGCCCA CGAUCAACAG AAGCAGAUAU 2220 UGGAAGUCGU AAACCGUCCAUCGGUGGCUU UGGAGCGAUC GUAGUGUGUC UGCAGGCGAU 2280 AACGACGUCU GUAAAGAGGCUAUUGACGAG GUAGGAGACG CUCUACUGCG CAAUUUGGAC 2340 CUUAACUUCC GCCGCAUGCGAUGCGGGACG AGCAGUUGGA UGGACCGCCA UGGAACCUGG 2400 AUCUGAAGCU UUCCAAACUGGUUCUGCGUG CGGUCCUUCC ACGUGGGAAG UGUUUGAUUC 2460 AUGGUUGCGG CGAGUCUUGCGGGCAAGACU CAGCUAUAAU UCCUCCAGAU CGAACUCCGU 2520 CUGCUUUCUU UCCCCCCGAUUGUACAAGAG UUGGAAGGCG ACGAAUAAAU CGGUCGCGGA 2580 AGCGGCAGAA AAGGGGUCUUGAAUAACAAG CCGUUAGGCA ACGAGCAGGG UGCUGACGGC 2640 GUCAACCCGA GCGGGGAAAUUGAGAUCGGU UCGAAGAAGA CGAAACGAAG CGCACGCUCC 2700 GGUCUCCAGC GGCCAAACGAGGAUGUCUUU GAGCCGUCAC GUUGCAGGGG GUUUCACUGA 2760 UGCAAGGCGA AGUGCAAGGGCGCAGCAGUC GAGAGGAAAA CCUGGAACGA GGAGGUCGUG 2820 AGGGAGGCAG AUGGCCACGGAGCCGGAGUA UAAACC 2856 2385 base pairs nucleic acid double lineargenomic DNA unknown 15 AAGCTTTTTT GCCTCTGCAA AAGTTCCTTT CTCGAATTGGTTTTTTGAGG AAAAGCAAGT 60 TAATAAACTA ATTATATTAT ATATAATTAG CAATTTTATAAAAAAAATAA AAAAATAGCC 120 CTGATTGCTG GCAACTGTGA GCTGAACATT GGTTAATCGGTCCATCTTTT TTTAAATATT 180 TTACATCGCT ACTTTTAAGT GCTTGACACT TGCATTTAATAGCTACTTTC TTTCCTTCAT 240 AAAAATTCCT TTTTTTTCCT TTAGTTTTCC GGTTAATTCCTTACGAAATT TTTTTCGTAC 300 GCTTCCCTTT TTTACTCTGA TAATTCTTTG AAGCAATGTCTGCTCTTTCG ACCTTAAAAA 360 AGCGCCTTGC TGCGTGTAAC CGAGCATCCC AATACAAGTTGGAAACAAGC TTAAACCCTA 420 TGCCTACATT TCGTTTGCTA CGCAATACGA AATGGTCATGGACACATTTG CAATATGTGT 480 TTCTAGCAGG TAATTTGATT TTTGCTTGTA TTGTCATTGAATCTCCTGGA TTCTGGGGGA 540 AATTTGGCAT TGCCTGTCTT TTGGCCATTG CGTTGACCGTTCCTTTAACA CGCCAAATTT 600 TTTTTCCTGC CATTGTTATC ATCACCTGGG CAATTTTATTTTACTCTTGT AGGTTTATTC 660 CAGAACGCTG GCGTCCACCC ATATGGGTTC GTGTTTTACCCACACTTGAA AATATTCTTT 720 ATGGCTCTAA TCTTTCTAGT CTTCTCTCGA AAACCACGCATAGCATCCTT GATATTTTGG 780 CCTGGGTTCC ATATGGAGTC ATGCATTATT CGGCTCCTTTTATCATTTCA TTTATTCTTT 840 TCATCTTTGC ACCTCCTGGA ACTCTTCCAG TTTGGGCTCGAACTTTTGGT TATATGAATT 900 TATTTGGTGT TCTTATCCAA ATGGCTTTCC CCTGTTCTCCTCCTTGGTAT GAAAATATGT 960 ATGGTTTAGA ACCTGCCACG TATGCAGTAC GTGGCTCTCCTGGTGGATTG GCCCGTATTG 1020 ATGCTCTCTT CGGCACTAGC ATTTACACTG ATTGTTTTTCTAACTCTCCG GTTGTTTTTG 1080 GTGCCTTTCC ATCTCTTCAC GCTGGATGGG CCATGCTGGAAGCACTTTTC CTTTCGCATG 1140 TGTTTCCTCG ATACCGCTTC TGCTTTTATG GATATGTTCTATGGCTTTGC TGGTGTACTA 1200 TGTACCTTAC CCACCACTAC TTTGTAGATT TGGTCGGCGGTATGTGTTTA GCTATTATAT 1260 GCTTCGTTTT TGCTCAAAAG CTACGCCTCC CACAGTTGCAAACTGGTAAA ATCCTTCGTT 1320 GGGAATACGA GTTTGTTATC CACGGTCATG GTCTTTCCGAAAAAACCAGC AACTCCTTGG 1380 CTCGTACCGG CAGCCCATAC TTACTTGGAA GGGATTCTTTTACTCAAAAC CCTAATGCAG 1440 TAGCCTTCAT GAGTGGTCTT AACAATATGG AACTTGCTAACACCGATCAT GAATGGTCCG 1500 TGGGTTCATC ATCACCTGAG CCGTTACCTA GTCCTGCTGCTGATTTGATT GATCGTCCTG 1560 CCAGTACCAC TTCCTCCATC TTTGATGCAA GTCATCTTCCTTAAATCAAC GTGCTTTAAG 1620 AATATATTTC CAAAAGCTAC ATGATACATT GACTAGAATCGGTTTGATTC ATAGTGGTAT 1680 TGGAATGATG TTGTTCATTG TGTTTTTTAA CTGTTAATCTGACATCCATT GAGTCATTCT 1740 TTACAATTTG TAAAATTAAT TTGTATCACT AATTTTGAAGGAAGCTATTT TGGTATTAAT 1800 ACCGCTTTTG GTCTCCACTT CCTTTTCGAA ACTCTTAACAGCGATTAGGC CGGGTATCTT 1860 CCAGTGTGAT GTATAGGTAT TTGTCGTTTT TTTATCATTTCCGTTAATAA AGAACTCTTT 1920 TATCCAGCTT CTTACACTGT CAACTGTTGT GAAAGGAACACATTTAGAAT TTCATTTTCC 1980 TTATTTGTTG TGATTTAAAT CGTTTGACAT AATTTTAAATTTGGTTTGAA ATGTGTGTGA 2040 GAAGGCTTGT TTTATTCATT TAGTTTATTG CTTGTTTGCACGAAAATCCA GAACGGAGCA 2100 TTAATGTAAT CCTTTTTTAT TCTGTAAAGC GTTTTTATACAAATGTTGGT TATACGTTTC 2160 TAAAATAAGA ATATTGTTAT AATAATATAG TTTTTTCTATCATTTGTTAC ACACACTAAA 2220 GAGACATTAA GGATAAGCAA ATGTGTTAAA ATGATAATATATTTTGGAAA CATTTATAAA 2280 GAAATTAAGC AGCTTTGACT AACTACATTT TTGTTTTTTTCCTAAGCAAA ACTGTATAGT 2340 TATACACGCG AGCTGTATTC ACTTCCATTG TAGTGACTTGAGCTC 2385 422 amino acids amino acid single linear peptide unknown 16Met Ser Ala Leu Ser Thr Leu Lys Lys Arg Leu Ala Ala Cys Asn 1 5 10 15Arg Ala Ser Gln Tyr Lys Leu Glu Thr Ser Leu Asn Pro Met Pro 20 25 30 ThrPhe Arg Leu Leu Arg Asn Thr Lys Trp Ser Trp Thr His Leu 35 40 45 Gln TyrVal Phe Leu Ala Gly Asn Leu Ile Phe Ala Cys Ile Val 50 55 60 Ile Glu SerPro Gly Phe Trp Gly Lys Phe Gly Ile Ala Cys Leu 65 70 75 Leu Ala Ile AlaLeu Thr Val Pro Leu Thr Arg Gln Ile Phe Phe 80 85 90 Pro Ala Ile Val IleIle Thr Trp Ala Ile Leu Phe Tyr Ser Cys 95 100 105 Arg Phe Ile Pro GluArg Trp Arg Pro Pro Ile Trp Val Arg Val 110 115 120 Leu Pro Thr Leu GluAsn Ile Leu Tyr Gly Ser Asn Leu Ser Ser 125 130 135 Leu Leu Ser Lys ThrThr His Ser Ile Leu Asp Ile Leu Ala Trp 140 145 150 Val Pro Tyr Gly ValMet His Tyr Ser Ala Pro Phe Ile Ile Ser 155 160 165 Phe Ile Leu Phe IlePhe Ala Pro Pro Gly Thr Leu Pro Val Trp 170 175 180 Ala Arg Thr Phe GlyTyr Met Asn Leu Phe Gly Val Leu Ile Gln 185 190 195 Met Ala Phe Pro CysSer Pro Pro Trp Tyr Glu Asn Met Tyr Gly 200 205 210 Leu Glu Pro Ala ThrTyr Ala Val Arg Gly Ser Pro Gly Gly Leu 215 220 225 Ala Arg Ile Asp AlaLeu Phe Gly Thr Ser Ile Tyr Thr Asp Cys 230 235 240 Phe Ser Asn Ser ProVal Val Phe Gly Ala Phe Pro Ser Leu His 245 250 255 Ala Gly Trp Ala MetLeu Glu Ala Leu Phe Leu Ser His Val Phe 260 265 270 Pro Arg Tyr Arg PheCys Phe Tyr Gly Tyr Val Leu Trp Leu Cys 275 280 285 Trp Cys Thr Met TyrLeu Thr His His Tyr Phe Val Asp Leu Val 290 295 300 Gly Gly Met Cys LeuAla Ile Ile Cys Phe Val Phe Ala Gln Lys 305 310 315 Leu Arg Leu Pro GlnLeu Gln Thr Gly Lys Ile Leu Arg Trp Glu 320 325 330 Tyr Glu Phe Val IleHis Gly His Gly Leu Ser Glu Lys Thr Ser 335 340 345 Asn Ser Leu Ala ArgThr Gly Ser Pro Tyr Leu Leu Gly Arg Asp 350 355 360 Ser Phe Thr Gln AsnPro Asn Ala Val Ala Phe Met Ser Gly Leu 365 370 375 Asn Asn Met Glu LeuAla Asn Thr Asp His Glu Trp Ser Val Gly 380 385 390 Ser Ser Ser Pro GluPro Leu Pro Ser Pro Ala Ala Asp Leu Ile 395 400 405 Asp Arg Pro Ala SerThr Thr Ser Ser Ile Phe Asp Ala Ser His 410 415 420 Leu Pro 2385 basepairs nucleic acid double linear genomic DNA unknown 17 AAGCTTTTTTGCCTCTGCAA AAGTTCCTTT CTCGAATTGG TTTTTTGAGG AAAAGCAAGT 60 TAATAAACTAATTATATTAT ATATAATTAG CAATTTTATA AAAAAAATAA AAAAATAGCC 120 CTGATTGCTGGCAACTGTGA GCTGAACATT GGTTAATCGG TCCATCTTTT TTTAAATATT 180 TTACATCGCTACTTTTAAGT GCTTGACACT TGCATTTAAT AGCTACTTTC TTTCCTTCAT 240 AAAAATTCCTTTTTTTTCCT TTAGTTTTCC GGTTAATTCC TTACGAAATT TTTTTCGTAC 300 GCTTCCCTTTTTTACTCTGA TAATTCTTTG AAGCAATGTC TGCTCTTTCG ACCTTAAAAA 360 AGCGCCTTGCTGCGTGTAAC CGAGCATCCC AATACAAGTT GGAAACAAGC TTAAACCCTA 420 TGCCTACATTTCGTTTGCTA CGCAATACGA AATGGTCATG GACACATTTG CAATATGTGT 480 TTCTAGCAGGTAATTTGATT TTTGCTTGTA TTGTCATTGA ATCTCCTGGA TTCTGGGGGA 540 AATTTGGCATTGCCTGTCTT TTGGCCATTG CGTTGACCGT TCCTTTAACA CGCCAAATTT 600 TTTTTCCTGCCATTGTTATC ATCACCTGGG CAATTTTATT TTACTCTTGT AGGTTTATTC 660 CAGAACGCTGGCGTCCACCC ATATGGGTTC GTGTTTTACC CACACTTGAA AATATTCTTT 720 ATGGCTCTAATCTTTCTAGT CTTCTCTCGA AAACCACGCA TAGCATCCTT GATATTTTGG 780 CCTGGGTTCCATATGGAGTC ATGCATTATT CGGCTCCTTT TATCATTTCA TTTATTCTTT 840 TCATCTTTGCACCTCCTGGA ACTCTTCCAG TTTGGGCTCG AACTTTTGGT TATATGAATT 900 TATTTGGTGTTCTTATCCAA ATGGCTTTCC CCTGTTCTCC TCCTTGGTAT GAAAATATGT 960 ATGGTTTAGAACCTGCCACG TATGCAGTAC GTGGCTCTCC TGGTGGATTG GCCCGTATTG 1020 ATGCTCTCTTCGGCACTAGC ATTTACACTG ATGGTTTTTC TAACTCTCCG GTTGTTTTTG 1080 GTGCCTTTCCATCTCTTCAC GCTGGATGGG CCATGCTGGA AGCACTTTTC CTTTCGCATG 1140 TGTTTCCTCGATACCGCTTC TGCTTTTATG GATATGTTCT ATGGCTTTGC TGGTGTACTA 1200 TGTACCTTACCCACCACTAC TTTGTAGATT TGGTCGGCGG TATGTGTTTA GCTATTATAT 1260 GCTTCGTTTTTGCTCAAAAG CTACGCCTCC CACAGTTGCA AACTGGTAAA ATCCTTCGTT 1320 GGGAATACGAGTTTGTTATC CACGGTCATG GTCTTTCCGA AAAAACCAGC AACTCCTTGG 1380 CTCGTACCGGCAGCCCATAC TTACTTGGAA GGGATTCTTT TACTCAAAAC CCTAATGCAG 1440 TAGCCTTCATGAGTGGTCTT AACAATATGG AACTTGCTAA CACCGATCAT GAATGGTCCG 1500 TGGGTTCATCATCACCTGAG CCGTTACCTA GTCCTGCTGC TGATTTGATT GATCGTCCTG 1560 CCAGTACCACTTCCTCCATC TTTGATGCAA GTCATCTTCC TTAAATCAAC GTGCTTTAAG 1620 AATATATTTCCAAAAGCTAC ATGATACATT GACTAGAATC GGTTTGATTC ATAGTGGTAT 1680 TGGAATGATGTTGTTCATTG TGTTTTTTAA CTGTTAATCT GACATCCATT GAGTCATTCT 1740 TTACAATTTGTAAAATTAAT TTGTATCACT AATTTTGAAG GAAGCTATTT TGGTATTAAT 1800 ACCGCTTTTGGTCTCCACTT CCTTTTCGAA ACTCTTAACA GCGATTAGGC CGGGTATCTT 1860 CCAGTGTGATGTATAGGTAT TTGTCGTTTT TTTATCATTT CCGTTAATAA AGAACTCTTT 1920 TATCCAGCTTCTTACACTGT CAACTGTTGT GAAAGGAACA CATTTAGAAT TTCATTTTCC 1980 TTATTTGTTGTGATTTAAAT CGTTTGACAT AATTTTAAAT TTGGTTTGAA ATGTGTGTGA 2040 GAAGGCTTGTTTTATTCATT TAGTTTATTG CTTGTTTGCA CGAAAATCCA GAACGGAGCA 2100 TTAATGTAATCCTTTTTTAT TCTGTAAAGC GTTTTTATAC AAATGTTGGT TATACGTTTC 2160 TAAAATAAGAATATTGTTAT AATAATATAG TTTTTTCTAT CATTTGTTAC ACACACTAAA 2220 GAGACATTAAGGATAAGCAA ATGTGTTAAA ATGATAATAT ATTTTGGAAA CATTTATAAA 2280 GAAATTAAGCAGCTTTGACT AACTACATTT TTGTTTTTTT CCTAAGCAAA ACTGTATAGT 2340 TATACACGCGAGCTGTATTC ACTTCCATTG TAGTGACTTG AGCTC 2385 422 amino acids amino acidsingle linear peptide unknown 18 Met Ser Ala Leu Ser Thr Leu Lys Lys ArgLeu Ala Ala Cys Asn 1 5 10 15 Arg Ala Ser Gln Tyr Lys Leu Glu Thr SerLeu Asn Pro Met Pro 20 25 30 Thr Phe Arg Leu Leu Arg Asn Thr Lys Trp SerTrp Thr His Leu 35 40 45 Gln Tyr Val Phe Leu Ala Gly Asn Leu Ile Phe AlaCys Ile Val 50 55 60 Ile Glu Ser Pro Gly Phe Trp Gly Lys Phe Gly Ile AlaCys Leu 65 70 75 Leu Ala Ile Ala Leu Thr Val Pro Leu Thr Arg Gln Ile PhePhe 80 85 90 Pro Ala Ile Val Ile Ile Thr Trp Ala Ile Leu Phe Tyr Ser Cys95 100 105 Arg Phe Ile Pro Glu Arg Trp Arg Pro Pro Ile Trp Val Arg Val110 115 120 Leu Pro Thr Leu Glu Asn Ile Leu Tyr Gly Ser Asn Leu Ser Ser125 130 135 Leu Leu Ser Lys Thr Thr His Ser Ile Leu Asp Ile Leu Ala Trp140 145 150 Val Pro Tyr Gly Val Met His Tyr Ser Ala Pro Phe Ile Ile Ser155 160 165 Phe Ile Leu Phe Ile Phe Ala Pro Pro Gly Thr Leu Pro Val Trp170 175 180 Ala Arg Thr Phe Gly Tyr Met Asn Leu Phe Gly Val Leu Ile Gln185 190 195 Met Ala Phe Pro Cys Ser Pro Pro Trp Tyr Glu Asn Met Tyr Gly200 205 210 Leu Glu Pro Ala Thr Tyr Ala Val Arg Gly Ser Pro Gly Gly Leu215 220 225 Ala Arg Ile Asp Ala Leu Phe Gly Thr Ser Ile Tyr Thr Asp Gly230 235 240 Phe Ser Asn Ser Pro Val Val Phe Gly Ala Phe Pro Ser Leu His245 250 255 Ala Gly Trp Ala Met Leu Glu Ala Leu Phe Leu Ser His Val Phe260 265 270 Pro Arg Tyr Arg Phe Cys Phe Tyr Gly Tyr Val Leu Trp Leu Cys275 280 285 Trp Cys Thr Met Tyr Leu Thr His His Tyr Phe Val Asp Leu Val290 295 300 Gly Gly Met Cys Leu Ala Ile Ile Cys Phe Val Phe Ala Gln Lys305 310 315 Leu Arg Leu Pro Gln Leu Gln Thr Gly Lys Ile Leu Arg Trp Glu320 325 330 Tyr Glu Phe Val Ile His Gly His Gly Leu Ser Glu Lys Thr Ser335 340 345 Asn Ser Leu Ala Arg Thr Gly Ser Pro Tyr Leu Leu Gly Arg Asp350 355 360 Ser Phe Thr Gln Asn Pro Asn Ala Val Ala Phe Met Ser Gly Leu365 370 375 Asn Asn Met Glu Leu Ala Asn Thr Asp His Glu Trp Ser Val Gly380 385 390 Ser Ser Ser Pro Glu Pro Leu Pro Ser Pro Ala Ala Asp Leu Ile395 400 405 Asp Arg Pro Ala Ser Thr Thr Ser Ser Ile Phe Asp Ala Ser His410 415 420 Leu Pro 2340 base pairs nucleic acid double linear genomicDNA unknown 19 TTTCTTTCTG TCAAAGAATA ATAAAGTGCC CATCAGTGTT CATATTTGTTACAAAGTGGT 60 TTTCTGATTT GGTACTACTG CAGAGGCGTA TTTTTTGCTT CAGTTACCATAGCGTAAGAA 120 CACTAGCGAC TTTTGTTCGT GAACCAACAG AGTAGGATTT CTACTGCTACATCTCTTAGG 180 TAGTTGGTTA GTCCGATCGC TCACTTTTGG TTGTTGTTAA GTACTTCATAAGTTTATCCT 240 TTTCCTTTTT CACACTGAGC TACTTTGGGT ATAGCTTTTG GCCCAAGGATCTTTGAATTT 300 TCTCCAAAAG TACTTTATTT TATATCCTAC AGGTTGCGGT TTTCATATTTTAAAAAGCTT 360 TTTAATCATT CCTTTGCGTA TGGCAAACCC TTTTTCGAGA TGGTTTCTATCAGAGAGACC 420 TCCAAACTGC CATGTAGCCG ATTTAGAAAC AAGTTTAGAT CCCCATCAAACGTTGTTGAA 480 GGTGCAAAAA TACAAACCCG CTTTAAGCGA CTGGGTGCAT TACATCTTCTTGGGATCCAT 540 CATGCTGTTT GTGTTCATTA CTAATCCCGC ACCTTGGATC TTCAAGATCCTTTTTTATTG 600 TTTCTTGGGC ACTTTATTCA TCATTCCAGC TACGTCACAG TTTTTCTTCAATGCCTTGCC 660 CATCCTAACA TGGGTGGCGC TGTATTTCAC TTCATCGTAC TTTCCAGATGACCGCAGGCC 720 TCCTATTACT GTCAAAGTGT TACCAGCGGT GGAAACAATT TTATACGGCGACAATTTAAG 780 TGATATTCTT GCAACATCGA CGAATTCCTT TTTGGACATT TTAGCATGGTTACCGTACGG 840 ACTATTTCAT TATGGGGCCC CATTTGTCGT TGCTGCCATC TTATTCGTATTTGGTCCACC 900 AACTGTTTTG CAAGGTTATG CTTTTGCATT TGGTTATATG AACCTGTTTGGTGTTATCAT 960 GCAAAATGTC TTTCCAGCCG CTCCCCCATG GTATAAAATT CTCTATGGATTGCAATCAGC 1020 CAACTATGAT ATGCATGGCT CGCCTGGTGG ATTAGCTAGA ATTGATAAGCTACTCGGTAT 1080 TAATATGTAT ACTACAGCTT TTTCAAATTC CTCCGTCATT TTCGGTGCTTTTCCTTCACT 1140 GCATTCCGGG TGTGCTACTA TGGAAGCCCT GTTTTTCTGT TATTGTTTTCCAAAATTGAA 1200 GCCCTTGTTT ATTGCTTATG TTTGCTGGTT ATGGTGGTCA ACTATGTATCTGACACACCA 1260 TTATTTTGTA GACCTTATGG CAGGTTCTGT GCTGTCATAC GTTATTTTCCAGTACACAAA 1320 GTACACACAT TTACCAATTG TAGATACATC TCTTTTTTGC AGATGGTCATACACTTCAAT 1380 TGAGAAATAC GATATATCAA AGAGTGATCC ATTGGCTGCA GATTCAAACGATATCGAAAG 1440 TGTCCCTTTG TCCAACTTGG AACTTGACTT TGATCTTAAT ATGACTGATGAACCCAGTGT 1500 AAGCCCTTCG TTATTTGATG GATCTACTTC TGTTTCTCGT TCGTCCGCCACGTCTATAAC 1560 GTCACTAGGT GTAAAGAGGG CTTAATGAGT ATTTTATCTG CAATTACGGATACGGTTGGT 1620 CTTATGTAGA TACATATAAA TATATATCTT TTTCTTTCTT TTTCTTAGTCAGGATTGTCG 1680 TTTAGCATAA TATACATGTA GTTTATTTAA TCACATACCA CTGATTATCTTTAGAATTTT 1740 ATAAATTTTT GAAATAAATG GGTGGCTTTT AATGGTGTCT ATGTTAAGTGAGGCTTTTAG 1800 AATGCTCTTC CTGCTTTGTT TATTATATGT GTATGAAAGA TATGTATGTATTTACATGTG 1860 TTTGTAGCGT CCCCAGTCAA AACCTGTGCG CTATACCTAA ATGGATTGATAATCTTCATT 1920 CACTAATTCT AAAATAGACT TCTTCCCCAA AGAACGGTGT AACGATGAGGCTCTATCCAG 1980 CTGCTTATCT AAATCAACTT TAACGATGGA TGATCTTATG ACACGGGGATCTTTCTTTAA 2040 AGTTCTTAGA ATTTCAGACT GTACCGCAGC TGATGAATCA AACAGCATTAAAAAGTGATA 2100 TGCTCGAAAA TGTTTTTCCT GGTCTTTCTT CATTATTTTA GGAAGATACCTTATGCCCAT 2160 GGGTACAATG TCCCTCACCA CACCTCTGTT TTGAATAATC AGTTTCCCGATTGTGGAAGA 2220 CAATTCTTTT GCTTCCAACT TTGGCGCATT GGAGTTGGTT ATGCGAACAAGTCCGATCAG 2280 CTCATAAAGC ATCTTAGTGA AAAGGGTGGT TTTGCGTTAT TCTTTCCTCTGTTGAAGCTT 2340 401 amino acids amino acid single linear peptide unknown20 Met Ala Asn Pro Phe Ser Arg Trp Phe Leu Ser Glu Arg Pro Pro 1 5 10 15Asn Cys His Val Ala Asp Leu Glu Thr Ser Leu Asp Pro His Gln 20 25 30 ThrLeu Leu Lys Val Gln Lys Tyr Lys Pro Ala Leu Ser Asp Trp 35 40 45 Val HisTyr Ile Phe Leu Gly Ser Ile Met Leu Phe Val Phe Ile 50 55 60 Thr Asn ProAla Pro Trp Ile Phe Lys Ile Leu Phe Tyr Cys Phe 65 70 75 Leu Gly Thr LeuPhe Ile Ile Pro Ala Thr Ser Gln Phe Phe Phe 80 85 90 Asn Ala Leu Pro IleLeu Thr Trp Val Ala Leu Tyr Phe Thr Ser 95 100 105 Ser Tyr Phe Pro AspAsp Arg Arg Pro Pro Ile Thr Val Lys Val 110 115 120 Leu Pro Ala Val GluThr Ile Leu Tyr Gly Asp Asn Leu Ser Asp 125 130 135 Ile Leu Ala Thr SerThr Asn Ser Phe Leu Asp Ile Leu Ala Trp 140 145 150 Leu Pro Tyr Gly LeuPhe His Tyr Gly Ala Pro Phe Val Val Ala 155 160 165 Ala Ile Leu Phe ValPhe Gly Pro Pro Thr Val Leu Gln Gly Tyr 170 175 180 Ala Phe Ala Phe GlyTyr Met Asn Leu Phe Gly Val Ile Met Gln 185 190 195 Asn Val Phe Pro AlaAla Pro Pro Trp Tyr Lys Ile Leu Tyr Gly 200 205 210 Leu Gln Ser Ala AsnTyr Asp Met His Gly Ser Pro Gly Gly Leu 215 220 225 Ala Arg Ile Asp LysLeu Leu Gly Ile Asn Met Tyr Thr Thr Ala 230 235 240 Phe Ser Asn Ser SerVal Ile Phe Gly Ala Phe Pro Ser Leu His 245 250 255 Ser Gly Cys Ala ThrMet Glu Ala Leu Phe Phe Cys Tyr Cys Phe 260 265 270 Pro Lys Leu Lys ProLeu Phe Ile Ala Tyr Val Cys Trp Leu Trp 275 280 285 Trp Ser Thr Met TyrLeu Thr His His Tyr Phe Val Asp Leu Met 290 295 300 Ala Gly Ser Val LeuSer Tyr Val Ile Phe Gln Tyr Thr Lys Tyr 305 310 315 Thr His Leu Pro IleVal Asp Thr Ser Leu Phe Cys Arg Trp Ser 320 325 330 Tyr Thr Ser Ile GluLys Tyr Asp Ile Ser Lys Ser Asp Pro Leu 335 340 345 Ala Ala Asp Ser AsnAsp Ile Glu Ser Val Pro Leu Ser Asn Leu 350 355 360 Glu Leu Asp Phe AspLeu Asn Met Thr Asp Glu Pro Ser Val Ser 365 370 375 Pro Ser Leu Phe AspGly Ser Thr Ser Val Ser Arg Ser Ser Ala 380 385 390 Thr Ser Ile Thr SerLeu Gly Val Lys Arg Ala 395 400 2340 base pairs nucleic acid doublelinear genomic DNA unknown 21 TTTCTTTCTG TCAAAGAATA ATAAAGTGCCCATCAGTGTT CATATTTGTT ACAAAGTGGT 60 TTTCTGATTT GGTACTACTG CAGAGGCGTATTTTTTGCTT CAGTTACCAT AGCGTAAGAA 120 CACTAGCGAC TTTTGTTCGT GAACCAACAGAGTAGGATTT CTACTGCTAC ATCTCTTAGG 180 TAGTTGGTTA GTCCGATCGC TCACTTTTGGTTGTTGTTAA GTACTTCATA AGTTTATCCT 240 TTTCCTTTTT CACACTGAGC TACTTTGGGTATAGCTTTTG GCCCAAGGAT CTTTGAATTT 300 TCTCCAAAAG TACTTTATTT TATATCCTACAGGTTGCGGT TTTCATATTT TAAAAAGCTT 360 TTTAATCATT CCTTTGCGTA TGGCAAACCCTTTTTCGAGA TGGTTTCTAT CAGAGAGACC 420 TCCAAACTGC CATGTAGCCG ATTTAGAAACAAGTTTAGAT CCCCATCAAA CGTTGTTGAA 480 GGTGCAAAAA TACAAACCCG CTTTAAGCGACTGGGTGCAT TACATCTTCT TGGGATCCAT 540 CATGCTGTTT GTGTTCATTA CTAATCCCGCACCTTGGATC TTCAAGATCC TTTTTTATTG 600 TTTCTTGGGC ACTTTATTCA TCATTCCAGCTACGTCACAG TTTTTCTTCA ATGCCTTGCC 660 CATCCTAACA TGGGTGGCGC TGTATTTCACTTCATCGTAC TTTCCAGATG ACCGCAGGCC 720 TCCTATTACT GTCAAAGTGT TACCAGCGGTGGAAACAATT TTATACGGCG ACAATTTAAG 780 TGATATTCTT GCAACATCGA CGAATTCCTTTTTGGACATT TTAGCATGGT TACCGTACGG 840 ACTATTTCAT TTTGGGGCCC CATTTGTCGTTGCTGCCATC TTATTCGTAT TTGGTCCACC 900 AACTGTTTTG CAAGGTTATG CTTTTGCATTTGGTTATATG AACCTGTTTG GTGTTATCAT 960 GCAAAATGTC TTTCCAGCCG CTCCCCCATGGTATAAAATT CTCTATGGAT TGCAATCAGC 1020 CAACTATGAT ATGCATGGCT CGCCTGGTGGATTAGCTAGA ATTGATAAGC TACTCGGTAT 1080 TAATATGTAT ACTACAGCTT TTTCAAATTCCTCCGTCATT TTCGGTGCTT TTCCTTCACT 1140 GCATTCCGGG TGTGCTACTA TGGAAGCCCTGTTTTTCTGT TATTGTTTTC CAAAATTGAA 1200 GCCCTTGTTT ATTGCTTATG TTTGCTGGTTATGGTGGTCA ACTATGTATC TGACACACCA 1260 TTATTTTGTA GACCTTATGG CAGGTTCTGTGCTGTCATAC GTTATTTTCC AGTACACAAA 1320 GTACACACAT TTACCAATTG TAGATACATCTCTTTTTTGC AGATGGTCAT ACACTTCAAT 1380 TGAGAAATAC GATATATCAA AGAGTGATCCATTGGCTGCA GATTCAAACG ATATCGAAAG 1440 TGTCCCTTTG TCCAACTTGG AACTTGACTTTGATCTTAAT ATGACTGATG AACCCAGTGT 1500 AAGCCCTTCG TTATTTGATG GATCTACTTCTGTTTCTCGT TCGTCCGCCA CGTCTATAAC 1560 GTCACTAGGT GTAAAGAGGG CTTAATGAGTATTTTATCTG CAATTACGGA TACGGTTGGT 1620 CTTATGTAGA TACATATAAA TATATATCTTTTTCTTTCTT TTTCTTAGTC AGGATTGTCG 1680 TTTAGCATAA TATACATGTA GTTTATTTAATCACATACCA CTGATTATCT TTAGAATTTT 1740 ATAAATTTTT GAAATAAATG GGTGGCTTTTAATGGTGTCT ATGTTAAGTG AGGCTTTTAG 1800 AATGCTCTTC CTGCTTTGTT TATTATATGTGTATGAAAGA TATGTATGTA TTTACATGTG 1860 TTTGTAGCGT CCCCAGTCAA AACCTGTGCGCTATACCTAA ATGGATTGAT AATCTTCATT 1920 CACTAATTCT AAAATAGACT TCTTCCCCAAAGAACGGTGT AACGATGAGG CTCTATCCAG 1980 CTGCTTATCT AAATCAACTT TAACGATGGATGATCTTATG ACACGGGGAT CTTTCTTTAA 2040 AGTTCTTAGA ATTTCAGACT GTACCGCAGCTGATGAATCA AACAGCATTA AAAAGTGATA 2100 TGCTCGAAAA TGTTTTTCCT GGTCTTTCTTCATTATTTTA GGAAGATACC TTATGCCCAT 2160 GGGTACAATG TCCCTCACCA CACCTCTGTTTTGAATAATC AGTTTCCCGA TTGTGGAAGA 2220 CAATTCTTTT GCTTCCAACT TTGGCGCATTGGAGTTGGTT ATGCGAACAA GTCCGATCAG 2280 CTCATAAAGC ATCTTAGTGA AAAGGGTGGTTTTGCGTTAT TCTTTCCTCT GTTGAAGCTT 2340 401 amino acids amino acid singlelinear peptide unknown 22 Met Ala Asn Pro Phe Ser Arg Trp Phe Leu SerGlu Arg Pro Pro 1 5 10 15 Asn Cys His Val Ala Asp Leu Glu Thr Ser LeuAsp Pro His Gln 20 25 30 Thr Leu Leu Lys Val Gln Lys Tyr Lys Pro Ala LeuSer Asp Trp 35 40 45 Val His Tyr Ile Phe Leu Gly Ser Ile Met Leu Phe ValPhe Ile 50 55 60 Thr Asn Pro Ala Pro Trp Ile Phe Lys Ile Leu Phe Tyr CysPhe 65 70 75 Leu Gly Thr Leu Phe Ile Ile Pro Ala Thr Ser Gln Phe Phe Phe80 85 90 Asn Ala Leu Pro Ile Leu Thr Trp Val Ala Leu Tyr Phe Thr Ser 95100 105 Ser Tyr Phe Pro Asp Asp Arg Arg Pro Pro Ile Thr Val Lys Val 110115 120 Leu Pro Ala Val Glu Thr Ile Leu Tyr Gly Asp Asn Leu Ser Asp 125130 135 Ile Leu Ala Thr Ser Thr Asn Ser Phe Leu Asp Ile Leu Ala Trp 140145 150 Leu Pro Tyr Gly Leu Phe His Phe Gly Ala Pro Phe Val Val Ala 155160 165 Ala Ile Leu Phe Val Phe Gly Pro Pro Thr Val Leu Gln Gly Tyr 170175 180 Ala Phe Ala Phe Gly Tyr Met Asn Leu Phe Gly Val Ile Met Gln 185190 195 Asn Val Phe Pro Ala Ala Pro Pro Trp Tyr Lys Ile Leu Tyr Gly 200205 210 Leu Gln Ser Ala Asn Tyr Asp Met His Gly Ser Pro Gly Gly Leu 215220 225 Ala Arg Ile Asp Lys Leu Leu Gly Ile Asn Met Tyr Thr Thr Ala 230235 240 Phe Ser Asn Ser Ser Val Ile Phe Gly Ala Phe Pro Ser Leu His 245250 255 Ser Gly Cys Ala Thr Met Glu Ala Leu Phe Phe Cys Tyr Cys Phe 260265 270 Pro Lys Leu Lys Pro Leu Phe Ile Ala Tyr Val Cys Trp Leu Trp 275280 285 Trp Ser Thr Met Tyr Leu Thr His His Tyr Phe Val Asp Leu Met 290295 300 Ala Gly Ser Val Leu Ser Tyr Val Ile Phe Gln Tyr Thr Lys Tyr 305310 315 Thr His Leu Pro Ile Val Asp Thr Ser Leu Phe Cys Arg Trp Ser 320325 330 Tyr Thr Ser Ile Glu Lys Tyr Asp Ile Ser Lys Ser Asp Pro Leu 335340 345 Ala Ala Asp Ser Asn Asp Ile Glu Ser Val Pro Leu Ser Asn Leu 350355 360 Glu Leu Asp Phe Asp Leu Asn Met Thr Asp Glu Pro Ser Val Ser 365370 375 Pro Ser Leu Phe Asp Gly Ser Thr Ser Val Ser Arg Ser Ser Ala 380385 390 Thr Ser Ile Thr Ser Leu Gly Val Lys Arg Ala 395 400 5340 basepairs nucleic acid double linear genomic DNA unknown 23 AGCGCTTCTATTTTCCTCCC CACCGCGAGG CGGAAATGGC ACATTTTTTT TCTTTTGCTT 60 CTGTGCTTTTGCTGTAATTT TTGGCATGTG CTATTGTATG AAGATAACGC GTGGTTCCGT 120 GGAAATAGCCGGAAATTTTG CCGGGAATAT GACGGACATG ATTTAACACC CGTGGAAATG 180 AAAAAAGCCAAGGTAAGAAA GTGGCAATAT TTTTCCTACA AATAGATCTG CTGTCCCTTA 240 GATGATTACCATACATATAT ATATTTATTA CACACATCTG TCAGAGGTAG CTAGCGAAGG 300 TGTCACTGAAATATTTTTTG TTCCAGTTAG TATAAATACG GAGGTAGAAC AGCTCTCCGC 360 GTGTATATCTTTTTTTGCGC TATACAAGAA CAGGAAGAAC GCATTTCCAT ACCTTTTTCT 420 CCTTACAGGTGCCCTCTGAG TAGTGTCACG AACGAGGAAA AAGATTAATA TTACTGTTTT 480 TATATTCAAAAAGAGTAAAG CCGTTGCTAT ATACGAATAT GACGATTACC GTGGGGGATG 540 CAGTTTCGGAGACGGAGCTG GAAAACAAAA GTCAAAACGT GGTACTATCT CCCAAGGCAT 600 CTGCTTCTTCAGACATAAGC ACAGATGTTG ATAAAGACAC ATCGTCTTCT TGGGATGACA 660 AATCTTTGCTGCCTACAGGT GAATATATTG TGGACAGAAA TAAGCCCCAA ACCTACTTGA 720 ATAGCGATGATATCGAAAAA GTGACAGAAT CTGATATTTT CCCTCAGAAA CGTCTGTTTT 780 CATTCTTGCACTCTAAGAAA ATTCCAGAAG TACCACAAAC CGATGACGAG AGGAAGATAT 840 ATCCTCTGTTCCATACAAAT ATTATCTCTA ACATGTTTTT TTGGTGGGTT CTACCCATCC 900 TGCGAGTTGGTTATAAGAGA ACGATACAGC CGAACGATCT CTTCAAAATG GATCCGAGGA 960 TGTCTATAGAGACCCTTTAT GACGACTTTG AAAAAAACAT GATTTACTAT TTTGAGAAGA 1020 CGAGGAAAAAATACCGTAAA AGACATCCAG AAGCGACAGA AGAAGAGGTT ATGGAAAATG 1080 CCAAACTACCTAAACATACA GTTCTGAGAG CTTTATTATT CACTTTTAAG AAACAGTACT 1140 TCATGTCGATAGTGTTTGCA ATTCTCGCTA ATTGTACATC CGGTTTTAAC CCCATGATTA 1200 CCAAGAGGCTAATTGAGTTT GTCGAAGAAA AGGCTATTTT TCATAGCATG CATGTTAACA 1260 AAGGTATTGGTTACGCTATT GGTGCATGTT TGATGATGTT CGTTAACGGG TTGACGTTCA 1320 ATCATTTCTTTCATACATCC CAACTGACTG GTGTGCAAGC TAAGTCTATT CTTACTAAAG 1380 CTGCCATGAAGAAAATGTTT AATGCATCTA ATTATGCGAG ACATTGTTTT CCTAACGGTA 1440 AAGTGACTTCTTTTGTAACA ACAGATCTCG CTAGAATTGA ATTTGCCTTA TCTTTTCAGC 1500 CGTTTTTGGCTGGGTTCCCT GCAATTTTGG CTATTTGCAT TGTTTTATTG ATCGTTAACC 1560 TTGGACCCATTGCCTTAGTT GGGATTGGTA TTTTTTTCGG TGGGTTTTTC ATATCCTTAT 1620 TTGCATTTAAGTTAATTCTG GGCTTTAGAA TTGCTGCGAA CATCTTCACT GATGCTAGAG 1680 TTACCATGATGAGAGAAGTG CTGAATAATA TAAAAATGAT TAAATATTAT ACGTGGGAGG 1740 ATGCGTATGAAAAAAATATT CAAGATATTA GGACCAAAGA GATTTCTAAA GTTAGAAAAA 1800 TGCAACTATCAAGAAATTTC TTGATTGCTA TGGCCATGTC TTTGCCTAGT ATTGCTTCAT 1860 TGGTCACTTTCCTTGCAATG TACAAAGTTA ATAAAGGAGG CAGGCAACCT GGTAATATTT 1920 TTGCCTCTTTATCTTTATTT CAGGTCTTGA GTTTGCAAAT GTTTTTCTTA CCTATTGCTA 1980 TTGGTACTGGAATTGACATG ATCATTGGAT TGGGCCGTTT GCAAAGCTTA TTGGAGGCTC 2040 CAGAAGATGATCCAAATCAG ATGATTGAAA TGAAGCCCTC TCCTGGCTTT GATCCAAAAT 2100 TGGCTCTAAAAATGACACAT TGCTCATTTG AGTGGGAAGA TTATGAATTA AACGACGCTA 2160 TTGAAGAAGCAAAAGGAGAA GCTAAAGATG AAGGTAAAAA GAACAAAAAA AAGCGTAAGG 2220 ATACATGGGGTAAGCCATCT GCAAGTACTA ATAAGGCGAA AAGATTGGAC AATATGTTGA 2280 AAGACAGAGACGGCCCGGAA GATTTAGAAA AAACTTCGTT TAGGGGTTTC AAGGACTTGA 2340 ACTTCGATATTAAAAAGGGC GAATTTATTA TGATTACGGG ACCTATTGGT ACTGGTAAAT 2400 CTTCATTATTGAATGCGATG GCAGGATCAA TGAGAAAAAT TGATGGTAAG GTTGAAGTCA 2460 ACGGGGACTTATTAATGTGT GGTTATCCAT GGATTCAAAA TGCATCTGTA AGAGATAACA 2520 TCATATTCGGTTCACCATTC AATAAAGAAA AGTATGATGA AGTAGTTCGT GTTTGCTCTT 2580 TGAAAGCTGATCTGGATATT TTACCGGCAG GCGATATGAC CGAAATTGGG GAACGTGGTA 2640 TTACTTTATCTGGTGGTCAA AAGGCACGTA TCAATTTAGC CAGGTCTGTT TATAAGAAGA 2700 AGGATATTTATGTATTCGAC GATGTCCTAA GTGCTGTCGA TTCTCGTGTT GGTAAACACA 2760 TCATGGATGAATGTCTAACC GGAATGCTTG CTAATAAAAC CAGAATTTTA GCAACGCATC 2820 AGTTGTCACTGATTGAGAGA GCTTCTAGAG TCATCGTTTT AGGTACTGAT GGCCAAGTCG 2880 ATATTGGTACTGTTGATGAG CTAAAAGCTC GTAATCAAAC TTTGATAAAT CTTTTACAAT 2940 TCTCTTCTCAAAATTCGGAG AAAGAGGATG AAGAACAGGA AGCGGTTGTT TCCGGTGAAT 3000 TGGGACAACTAAAATATGAA CCAGAGGTAA AGGAATTGAC TGAACTGAAG AAAAAGGCTA 3060 CAGAAATGTCACAAACTGCA AATAGTGGTA AAATTGTAGC GGATGGTCAT ACTAGTAGTA 3120 AAGAAGAAAGAGCAGTCAAT AGTATCAGTC TGAAAATATA CCGTGAATAC ATTAAAGCTG 3180 CAGTAGGTAAGTGGGGTTTT ATCGCACTAC CGTTGTATGC AATTTTAGTC GTTGGAACCA 3240 CATTCTGCTCACTTTTTTCT TCCGTTTGGT TATCTTACTG GACTGAGAAT AAATTCAAAA 3300 ACAGACCACCCAGTTTTTAT ATGGGTCTTT ACTCCTTCTT TGTGTTTGCT GCTTTCATAT 3360 TCATGAATGGCCAGTTCACC ATACTTTGCG CAATGGGTAT TATGGCATCG AAATGGTTAA 3420 ATTTGAGGGCTGTGAAAAGA ATTTTACACA CTCCAATGTC ATACATAGAT ACCACACCTT 3480 TGGGACGTATTCTGAACAGA TTCACAAAAG ATACAGATAG CTTAGATAAT GAGTTAACCG 3540 AAAGTTTACGGTTGATGACA TCTCAATTTG CTAATATTGT AGGTGTTTGC GTCATGTGTA 3600 TTGTTTACTTGCCGTGGTTT GCTATCGCAA TTCCGTTTCT TTTGGTCATC TTTGTTCTGA 3660 TTGCTGATCATTATCAGAGT TCTGGTAGAG AAATTAAAAG ACTTGAAGCT GTGCAACGGT 3720 CTTTTGTTTACAATAATTTA AATGAAGTTT TGGGTGGGAT GGATACAATC AAAGCATACC 3780 GAAGTCAGGAACGATTTTTG GCGAAATCAG ATTTTTTGAT CAACAAGATG AATGAGGCGG 3840 GATACCTTGTAGTTGTCCTG CAAAGATGGG TAGGTATTTT CCTTGATATG GTTGCTATCG 3900 CATTTGCACTAATTATTACG TTATTGTGTG TTACGAGAGC CTTTCCTATT TCCGCGGCTT 3960 CAGTTGGTGTTTTGTTGACT TATGTATTAC AATTGCCTGG TCTATTAAAT ACCATTTTAA 4020 GGGCAATGACTCAAACAGAG AATGACATGA ATAGTGCCGA AAGATTGGTA ACATATGCAA 4080 CTGAACTACCACTAGAGGCA TCCTATAGAA AGCCCGAAAT GACACCTCCA GAGTCATGGC 4140 CCTCAATGGGCGAAATAATT TTTGAAAATG TTGATTTTGC CTATAGACCT GGTTTACCTA 4200 TAGTTTTAAAAAATCTTAAC TTGAATATCA AGAGTGGGGA AAAAATTGGT ATCTGTGGTC 4260 GTACAGGTGCTGGTAAGTCC ACTATTATGA GTGCCCTTTA CAGGTTGAAT GAATTGACCG 4320 CAGGTAAAATTTTAATTGAC AATGTTGATA TAAGTCAGCT GGGACTTTTC GATTTAAGAA 4380 GAAAATTAGCCATCATTCCA CAAGATCCAG TATTATTTAG GGGTACGATT CGCAAGAACT 4440 TAGATCCATTTAATGAGCGT ACAGATGACG AATTATGGGA TGCATTGGTG AGAGGTGGTG 4500 CTATCGCCAAGGATGACTTG CCGGAAGTGA AATTGCAAAA ACCTGATGAA AATGGTACTC 4560 ATGGTAAAATGCATAAGTTC CATTTAGATC AAGCAGTGGA AGAAGAGGGC TCCAATTTCT 4620 CCTTAGGTGAGAGACAACTA TTAGCATTAA CAAGGGCATT GGTCCGCCAA TCAAAAATAT 4680 TGATTTTGGATGAGGCTACA TCCTCAGTGG ACTACGAAAC GGATGGCAAA ATCCAAACAC 4740 GTATTGTTGAGGAATTTGGA GATTGTACAA TTTTGTGTAT TGCTCACAGA CTGAAGACCA 4800 TTGTAAATTATGATCGTATT CTTGTTTTAG AGAAGGGTGA AGTCGCAGAA TTCGATACAC 4860 CATGGACGTTGTTTAGTCAA GAAGATAGTA TTTTCAGAAG CATGTGTTCT AGATCTGGTA 4920 TTGTGGAAAATGATTTCGAG AACAGAAGTT AATTTATATT ATTTGTTGCA TGATTTTTCT 4980 CTTTTATTTATTTATATGTT GCCGATGGTA CAAATTAGTA CTAGAAAAGA AAACCCACTA 5040 CTATGACTTGCAGAAAAAGT TATGTGTGCC ATAGATAGAT ATAATTGCAT ACCCACATCG 5100 TATACTCAAAATTCCGAAAA GAACATTTCA TTTTTTATGA GGCAAACTGA ACAACGCTTC 5160 GGTCCTTTTTTCATTCTAGA AATATATATT TATACATCAT TTTCAGAAGA TATTCAAAGA 5220 ACTTATTGGGATGTCTATTT ACTGAATAAA GTATACACAA AAAACGAATT TAAAATGGAA 5280 GGCATAAATAGAAAACTTAG AAGTGAAAAT CCTAAAACCG AAGGATATTT CAAATACGTA 5340 1477 aminoacids amino acid single linear peptide unknown 24 Met Thr Ile Thr ValGly Asp Ala Val Ser Glu Thr Glu Leu Glu 5 10 15 Asn Lys Ser Gln Asn ValVal Leu Ser Pro Lys Ala Ser Ala Ser 20 25 30 Ser Asp Ile Ser Thr Asp ValAsp Lys Asp Thr Ser Ser Ser Trp 35 40 45 Asp Asp Lys Ser Leu Leu Pro ThrGly Glu Tyr Ile Val Asp Arg 50 55 60 Asn Lys Pro Gln Thr Tyr Leu Asn SerAsp Asp Ile Glu Lys Val 65 70 75 Thr Glu Ser Asp Ile Phe Pro Gln Lys ArgLeu Phe Ser Phe Leu 80 85 90 His Ser Lys Lys Ile Pro Glu Val Pro Gln ThrAsp Asp Glu Arg 95 100 105 Lys Ile Tyr Pro Leu Phe His Thr Asn Ile IleSer Asn Met Phe 110 115 120 Phe Trp Trp Val Leu Pro Ile Leu Arg Val GlyTyr Lys Arg Thr 125 130 135 Ile Gln Pro Asn Asp Leu Phe Lys Met Asp ProArg Met Ser Ile 140 145 150 Glu Thr Leu Tyr Asp Asp Phe Glu Lys Asn MetIle Tyr Tyr Phe 155 160 165 Glu Lys Thr Arg Lys Lys Tyr Arg Lys Arg HisPro Glu Ala Thr 170 175 180 Glu Glu Glu Val Met Glu Asn Ala Lys Leu ProLys His Thr Val 185 190 195 Leu Arg Ala Leu Leu Phe Thr Phe Lys Lys GlnTyr Phe Met Ser 200 205 210 Ile Val Phe Ala Ile Leu Ala Asn Cys Thr SerGly Phe Asn Pro 215 220 225 Met Ile Thr Lys Arg Leu Ile Glu Phe Val GluGlu Lys Ala Ile 230 235 240 Phe His Ser Met His Val Asn Lys Gly Ile GlyTyr Ala Ile Gly 245 250 255 Ala Cys Leu Met Met Phe Val Asn Gly Leu ThrPhe Asn His Phe 260 265 270 Phe His Thr Ser Gln Leu Thr Gly Val Gln AlaLys Ser Ile Leu 275 280 285 Thr Lys Ala Ala Met Lys Lys Met Phe Asn AlaSer Asn Tyr Ala 290 295 300 Arg His Cys Phe Pro Asn Gly Lys Val Thr SerPhe Val Thr Thr 305 310 315 Asp Leu Ala Arg Ile Glu Phe Ala Leu Ser PheGln Pro Phe Leu 320 325 330 Ala Gly Phe Pro Ala Ile Leu Ala Ile Cys IleVal Leu Leu Ile 335 340 345 Val Asn Leu Gly Pro Ile Ala Leu Val Gly IleGly Ile Phe Phe 350 355 360 Gly Gly Phe Phe Ile Ser Leu Phe Ala Phe LysLeu Ile Leu Gly 365 370 375 Phe Arg Ile Ala Ala Asn Ile Phe Thr Asp AlaArg Val Thr Met 380 385 390 Met Arg Glu Val Leu Asn Asn Ile Lys Met IleLys Tyr Tyr Thr 395 400 405 Trp Glu Asp Ala Tyr Glu Lys Asn Ile Gln AspIle Arg Thr Lys 410 415 420 Glu Ile Ser Lys Val Arg Lys Met Gln Leu SerArg Asn Phe Leu 425 430 435 Ile Ala Met Ala Met Ser Leu Pro Ser Ile AlaSer Leu Val Thr 440 445 450 Phe Leu Ala Met Tyr Lys Val Asn Lys Gly GlyArg Gln Pro Gly 455 460 465 Asn Ile Phe Ala Ser Leu Ser Leu Phe Gln ValLeu Ser Leu Gln 470 475 480 Met Phe Phe Leu Pro Ile Ala Ile Gly Thr GlyIle Asp Met Ile 485 490 495 Ile Gly Leu Gly Arg Leu Gln Ser Leu Leu GluAla Pro Glu Asp 500 505 510 Asp Pro Asn Gln Met Ile Glu Met Lys Pro SerPro Gly Phe Asp 515 520 525 Pro Lys Leu Ala Leu Lys Met Thr His Cys SerPhe Glu Trp Glu 530 535 540 Asp Tyr Glu Leu Asn Asp Ala Ile Glu Glu AlaLys Gly Glu Ala 545 550 555 Lys Asp Glu Gly Lys Lys Asn Lys Lys Lys ArgLys Asp Thr Trp 560 565 570 Gly Lys Pro Ser Ala Ser Thr Asn Lys Ala LysArg Leu Asp Asn 575 580 585 Met Leu Lys Asp Arg Asp Gly Pro Glu Asp LeuGlu Lys Thr Ser 590 595 600 Phe Arg Gly Phe Lys Asp Leu Asn Phe Asp IleLys Lys Gly Glu 605 610 615 Phe Ile Met Ile Thr Gly Pro Ile Gly Thr GlyLys Ser Ser Leu 620 625 630 Leu Asn Ala Met Ala Gly Ser Met Arg Lys IleAsp Gly Lys Val 635 640 645 Glu Val Asn Gly Asp Leu Leu Met Cys Gly TyrPro Trp Ile Gln 650 655 660 Asn Ala Ser Val Arg Asp Asn Ile Ile Phe GlySer Pro Phe Asn 665 670 675 Lys Glu Lys Tyr Asp Glu Val Val Arg Val CysSer Leu Lys Ala 680 685 690 Asp Leu Asp Ile Leu Pro Ala Gly Asp Met ThrGlu Ile Gly Glu 695 700 705 Arg Gly Ile Thr Leu Ser Gly Gly Gln Lys AlaArg Ile Asn Leu 710 715 720 Ala Arg Ser Val Tyr Lys Lys Lys Asp Ile TyrVal Phe Asp Asp 725 730 735 Val Leu Ser Ala Val Asp Ser Arg Val Gly LysHis Ile Met Asp 740 745 750 Glu Cys Leu Thr Gly Met Leu Ala Asn Lys ThrArg Ile Leu Ala 755 760 765 Thr His Gln Leu Ser Leu Ile Glu Arg Ala SerArg Val Ile Val 770 775 780 Leu Gly Thr Asp Gly Gln Val Asp Ile Gly ThrVal Asp Glu Leu 785 790 795 Lys Ala Arg Asn Gln Thr Leu Ile Asn Leu LeuGln Phe Ser Ser 800 805 810 Gln Asn Ser Glu Lys Glu Asp Glu Glu Gln GluAla Val Val Ser 815 820 825 Gly Glu Leu Gly Gln Leu Lys Tyr Glu Pro GluVal Lys Glu Leu 830 835 840 Thr Glu Leu Lys Lys Lys Ala Thr Glu Met SerGln Thr Ala Asn 845 850 855 Ser Gly Lys Ile Val Ala Asp Gly His Thr SerSer Lys Glu Glu 860 865 870 Arg Ala Val Asn Ser Ile Ser Leu Lys Ile TyrArg Glu Tyr Ile 875 880 885 Lys Ala Ala Val Gly Lys Trp Gly Phe Ile AlaLeu Pro Leu Tyr 890 895 900 Ala Ile Leu Val Val Gly Thr Thr Phe Cys SerLeu Phe Ser Ser 905 910 915 Val Trp Leu Ser Tyr Trp Thr Glu Asn Lys PheLys Asn Arg Pro 920 925 930 Pro Ser Phe Tyr Met Gly Leu Tyr Ser Phe PheVal Phe Ala Ala 935 940 945 Phe Ile Phe Met Asn Gly Gln Phe Thr Ile LeuCys Ala Met Gly 950 955 960 Ile Met Ala Ser Lys Trp Leu Asn Leu Arg AlaVal Lys Arg Ile 965 970 975 Leu His Thr Pro Met Ser Tyr Ile Asp Thr ThrPro Leu Gly Arg 980 985 990 Ile Leu Asn Arg Phe Thr Lys Asp Thr Asp SerLeu Asp Asn Glu 995 1000 1005 Leu Thr Glu Ser Leu Arg Leu Met Thr SerGln Phe Ala Asn Ile 1010 1015 1020 Val Gly Val Cys Val Met Cys Ile ValTyr Leu Pro Trp Phe Ala 1025 1030 1035 Ile Ala Ile Pro Phe Leu Leu ValIle Phe Val Leu Ile Ala Asp 1040 1045 1050 His Tyr Gln Ser Ser Gly ArgGlu Ile Lys Arg Leu Glu Ala Val 1055 1060 1065 Gln Arg Ser Phe Val TyrAsn Asn Leu Asn Glu Val Leu Gly Gly 1070 1075 1080 Met Asp Thr Ile LysAla Tyr Arg Ser Gln Glu Arg Phe Leu Ala 1085 1090 1095 Lys Ser Asp PheLeu Ile Asn Lys Met Asn Glu Ala Gly Tyr Leu 1100 1105 1110 Val Val ValLeu Gln Arg Trp Val Gly Ile Phe Leu Asp Met Val 1115 1120 1125 Ala IleAla Phe Ala Leu Ile Ile Thr Leu Leu Cys Val Thr Arg 1130 1135 1140 AlaPhe Pro Ile Ser Ala Ala Ser Val Gly Val Leu Leu Thr Tyr 1145 1150 1155Val Leu Gln Leu Pro Gly Leu Leu Asn Thr Ile Leu Arg Ala Met 1160 11651170 Thr Gln Thr Glu Asn Asp Met Asn Ser Ala Glu Arg Leu Val Thr 11751180 1185 Tyr Ala Thr Glu Leu Pro Leu Glu Ala Ser Tyr Arg Lys Pro Glu1190 1195 1200 Met Thr Pro Pro Glu Ser Trp Pro Ser Met Gly Glu Ile IlePhe 1205 1210 1215 Glu Asn Val Asp Phe Ala Tyr Arg Pro Gly Leu Pro IleVal Leu 1220 1225 1230 Lys Asn Leu Asn Leu Asn Ile Lys Ser Gly Glu LysIle Gly Ile 1235 1240 1245 Cys Gly Arg Thr Gly Ala Gly Lys Ser Thr IleMet Ser Ala Leu 1250 1255 1260 Tyr Arg Leu Asn Glu Leu Thr Ala Gly LysIle Leu Ile Asp Asn 1265 1270 1275 Val Asp Ile Ser Gln Leu Gly Leu PheAsp Leu Arg Arg Lys Leu 1280 1285 1290 Ala Ile Ile Pro Gln Asp Pro ValLeu Phe Arg Gly Thr Ile Arg 1295 1300 1305 Lys Asn Leu Asp Pro Phe AsnGlu Arg Thr Asp Asp Glu Leu Trp 1310 1315 1320 Asp Ala Leu Val Arg GlyGly Ala Ile Ala Lys Asp Asp Leu Pro 1325 1330 1335 Glu Val Lys Leu GlnLys Pro Asp Glu Asn Gly Thr His Gly Lys 1340 1345 1350 Met His Lys PheHis Leu Asp Gln Ala Val Glu Glu Glu Gly Ser 1355 1360 1365 Asn Phe SerLeu Gly Glu Arg Gln Leu Leu Ala Leu Thr Arg Ala 1370 1375 1380 Leu ValArg Gln Ser Lys Ile Leu Ile Leu Asp Glu Ala Thr Ser 1385 1390 1395 SerVal Asp Tyr Glu Thr Asp Gly Lys Ile Gln Thr Arg Ile Val 1400 1405 1410Glu Glu Phe Gly Asp Cys Thr Ile Leu Cys Ile Ala His Arg Leu 1415 14201425 Lys Thr Ile Val Asn Tyr Asp Arg Ile Leu Val Leu Glu Lys Gly 14301435 1440 Glu Val Ala Glu Phe Asp Thr Pro Trp Thr Leu Phe Ser Gln Glu1445 1450 1455 Asp Ser Ile Phe Arg Ser Met Cys Ser Arg Ser Gly Ile ValGlu 1460 1465 1470 Asn Asp Phe Glu Asn Arg Ser 1475 26 bases nucleicacid single linear Other nucleic acid (synthetic DNA) unknown 25TTTGGTTAYA TGAAYYTNTT YGGNGT 26 29 bases nucleic acid single linearOther nucleic acid (synthetic DNA) unknown 26 TCTACAAART ARTGGTGNGTNARRTACAT 29 2274 base pairs nucleic acid double linear genomic DNAunknown 27 TTATATATAT TATTGATTTG TTCCTGTTGT TATTTAGTTT AGAATCAGACGACTACACCA 60 GAACCACAAT TCAACCAACA CTTATATAGA ACCTGGCTTG GAAAAAAGTAACATTTATCA 120 TTCCTATACT TTTTTAGCAA ACATAATCCG TGTTTTACAT ATATTATTCACCCAATATCA 180 TAACAAAAAC AAACTGAATA ATGGCGTCTT CTATTTTGCG TTCCAAAATAATACAAAAAC 240 CGTACCAATT ATTCCACTAC TATTTTCTTC TGGAGAAGGC TCCTGGTTCTACAGTTAGTG 300 ATTTGAATTT TGATACAAAC ATACAAACGA GTTTACGTAA ATTAAAGCATCATCATTGGA 360 CGGTGGGAGA AATATTCCAT TATGGGTTTT TGGTTTCCAT ACTTTTTTTCGTGTTTGTGG 420 TTTTCCCAGC TTCATTTTTT ATAAAATTAC CAATAATCTT AGCATTTGCTACTTGTTTTT 480 TAATACCCTT AACATCACAA TTTTTTCTTC CTGCCTTGCC CGTTTTCACTTGGTTGGCAT 540 TATATTTTAC GTGTGCTAAA ATACCTCAAG AATGGAAACC AGCTATCACAGTTAAAGTTT 600 TACCAGCTAT GGAAACAATT TTGTACGGCG ATAATTTATC AAATGTTTTGGCAACCATCA 660 CTACCGGAGT GTTAGATATA TTGGCATGGT TACCATATGG GATTATTCATTTCAGTTTCC 720 CATTTGTACT TGCTGCTATT ATATTTTTAT TTGGGCCACC GACGGCATTAAGATCATTTG 780 GATTTGCCTT TGGTTATATG AACTTGCTTG GAGTCTTGAT TCAAATGGCATTCCCAGCTG 840 CTCCTCCATG GTACAAAAAC TTGCACGGAT TAGAACCAGC TAATTATTCAATGCACGGGT 900 CTCCTGGTGG ACTTGGAAGG ATAGATAAAT TGTTAGGTGT TGATATGTATACCACAGGGT 960 TTTCCAATTC ATCAATCATT TTTGGGGCAT TCCCATCGTT ACATTCAGGATGTTGTATCA 1020 TGGAAGTGTT ATTTTTGTGT TGGTTGTTTC CACGATTCAA GTTTGTGTGGGTTACATACG 1080 CATCTTGGCT TTGGTGGAGC ACGATGTATT TGACCCATCA CTACTTTGTCGATTTGATTG 1140 GTGGAGCCAT GCTATCTTTG ACTGTTTTTG AGTTCACCAA ATATAAATATTTGCCAAAAA 1200 ACAAAGAAGG CCTTTTCTGT CGTTGGTCAT ACACTGAAAT TGAAAAAATCGATATCCAAG 1260 AGATTGACCC TTTATCATAC AATTATATCC CTGTCAACAG CAATGATAATGAAAGCAGAT 1320 TGTATACGAG AGTGTACCAA GAGTCTCAGG TTAGTCCCCC ACAGAGAGCTGAAACACCTG 1380 AAGCATTTGA GATGTCAAAT TTTTCTAGGT CTAGACAAAG CTCAAAGACTCAGGTTCCAT 1440 TGAGTAATCT TACTAACAAT GATCAAGTGT CTGGAATTAA CGAAGAGGATGAAGAAGAAG 1500 AAGGCGATGA AATTTCATCG AGTACTCCTT CGGTGTTTGA AGACGAACCACAGGGTAGCA 1560 CATATGCTGC ATCCTCAGCT ACATCAGTAG ATGATTTGGA TTCCAAAAGAAATTAGTAAA 1620 ATAACAGTTT CTATTAATTT CTTTATTTCC TCCTAATTAA TGATTTTATGCTCAATACCT 1680 ACACTATCTG TTTTTAATTT CCTACTTTTT TTTTATTATT GTTGAGTTCATTTGCTGTTC 1740 ATTGAATATT TACAATTTTG CATTAATTAC CATCAATATA GAATGGGCACAGTTTTTTTA 1800 AGTTTTTTTG TTTTTGTGTT TGTCTTTCTT TTTTTACATT AATGTGTTTGGATTGTTTTA 1860 GGTTCCTTTA TCCCTTAGCC CCCTCAGAAT ACTATTTTAT CTAATTAATTTGTTTTTATT 1920 TTCTGATATT TACCAATTGC TTTTTCTTTT GGATATTTAT AATAGCATCCCCTAATAATT 1980 AATATACAAC TGTTTCATAT ATATACGTGT ATGTCCTGTA GTGGTGGAAACTGGAGTCAA 2040 CATTTGTATT AATGTGTACA AGAAAGCAGT GTTAATGCTA CTATTATAATTTTTGAGGTG 2100 CAAATCAAGA GGTTGGCAGC TTTCTTATGG CTATGACCGT GAATGAAGGCTTGTAAACCA 2160 CGTAATAAAC AAAAGCCAAC AAGTTTTTTT AGAGCCTTTA ACAACATACGCAATGAGAGT 2220 GATTGCAATA CTACAAGATA TAGCCCAAAA AATTGAATGC ATTTCAACAACAAC 2274 471 amino acids amino acid single linear peptide unknown 28Met Ala Ser Ser Ile Leu Arg Ser Lys Ile Ile Gln Lys Pro Tyr 5 10 15 GlnLeu Phe His Tyr Tyr Phe Leu Ser Glu Lys Ala Pro Gly Ser 20 25 30 Thr ValSer Asp Leu Asn Phe Asp Thr Asn Ile Gln Thr Ser Leu 35 40 45 Arg Lys LeuLys His His His Trp Thr Val Gly Glu Ile Phe His 50 55 60 Tyr Gly Phe LeuVal Ser Ile Leu Phe Phe Val Phe Val Val Phe 65 70 75 Pro Ala Ser Phe PheIle Lys Leu Pro Ile Ile Leu Ala Phe Ala 80 85 90 Thr Cys Phe Leu Ile ProLeu Thr Ser Gln Phe Phe Leu Pro Ala 95 100 105 Leu Pro Val Phe Thr TrpLeu Ala Leu Tyr Phe Thr Cys Ala Lys 110 115 120 Ile Pro Gln Glu Trp LysPro Ala Ile Thr Val Lys Val Leu Pro 125 130 135 Ala Met Glu Thr Ile LeuTyr Gly Asp Asn Leu Ser Asn Val Leu 140 145 150 Ala Thr Ile Thr Thr GlyVal Leu Asp Ile Leu Ala Trp Leu Pro 155 160 165 Tyr Gly Ile Ile His PheSer Phe Pro Phe Val Leu Ala Ala Ile 170 175 180 Ile Phe Leu Phe Gly ProPro Thr Ala Leu Arg Ser Phe Gly Phe 185 190 195 Ala Phe Gly Tyr Met AsnLeu Leu Gly Val Leu Ile Gln Met Ala 200 205 210 Phe Pro Ala Ala Pro ProTrp Tyr Lys Asn Leu His Gly Leu Glu 215 220 225 Pro Ala Asn Tyr Ser MetHis Gly Ser Pro Gly Gly Leu Gly Arg 230 235 240 Ile Asp Lys Leu Leu GlyVal Asp Met Tyr Thr Thr Gly Phe Ser 245 250 255 Asn Ser Ser Ile Ile PheGly Ala Phe Pro Ser Leu His Ser Gly 260 265 270 Cys Cys Ile Met Glu ValLeu Phe Leu Cys Trp Leu Phe Pro Arg 275 280 285 Phe Lys Phe Val Trp ValThr Tyr Ala Ser Trp Leu Trp Trp Ser 290 295 300 Thr Met Tyr Leu Thr HisHis Tyr Phe Val Asp Leu Ile Gly Gly 305 310 315 Ala Met Leu Ser Leu ThrVal Phe Glu Phe Thr Lys Tyr Lys Tyr 320 325 330 Leu Pro Lys Asn Lys GluGly Leu Phe Cys Arg Trp Ser Tyr Thr 335 340 345 Glu Ile Glu Lys Ile AspIle Gln Glu Ile Asp Pro Leu Ser Tyr 350 355 360 Asn Tyr Ile Pro Val AsnSer Asn Asp Asn Glu Ser Arg Leu Tyr 365 370 375 Thr Arg Val Tyr Gln GluSer Gln Val Ser Pro Pro Gln Arg Ala 380 385 390 Glu Thr Pro Glu Ala PheGlu Met Ser Asn Phe Ser Arg Ser Arg 395 400 405 Gln Ser Ser Lys Thr GlnVal Pro Leu Ser Asn Leu Thr Asn Asn 410 415 420 Asp Gln Val Ser Gly IleAsn Glu Glu Asp Glu Glu Glu Glu Gly 425 430 435 Asp Glu Ile Ser Ser SerThr Pro Ser Val Phe Glu Asp Glu Pro 440 445 450 Gln Gly Ser Thr Tyr AlaAla Ser Ser Ala Thr Ser Val Asp Asp 455 460 465 Leu Asp Ser Lys Arg Asn470 243 base pairs nucleic acid double linear genomic DNA unknown 29TTTGAAAAAT TTGAATTTTA AAATTAATCC AATGGAAAAA ATTGGTATTT GTGGAAGAAC 60CGGTGCTGGT AAATCATCAA TTATGACAGC ATTATATCGA TTATCAGAAT TAGAACTGGG 120GAAAATTATT ATTGATGATA TTGATATTTC AACTTTGGGT TTAAAAGATC TTCGATCAAA 180ATTATCAATT ATTCCTCAAG ATCCAGTATT ATTCCGAGGT TCAATTCGGA AAAACTTGGA 240TCC 243 80 amino acids amino acid single linear peptide unknown 30 LeuLys Asn Leu Asn Phe Lys Ile Asn Pro Met Glu Lys Ile Gly 5 10 15 Ile CysGly Arg Thr Gly Ala Gly Lys Ser Ser Ile Met Thr Ala 20 25 30 Leu Tyr ArgLeu Ser Glu Leu Glu Leu Gly Lys Ile Ile Ile Asp 35 40 45 Asp Ile Asp IleSer Thr Leu Gly Leu Lys Asp Leu Arg Ser Lys 50 55 60 Leu Ser Ile Ile ProGln Asp Pro Val Leu Phe Arg Gly Ser Ile 65 70 75 Arg Lys Asn Leu Asp 801601 bases nucleic acid double linear genomic DNA Yes unknown 31AGGAAGATGA CTTGCATCAA AGATGGAGGA AGTGGTACTG GCAGGACGAT CAATCAAATC 60AGCAGCAGGA CTAGGTAACG GCTCAGGTGA TGATGAACCC ACGGACCATT CATGATCGGT 120GTTAGCAAGT TCCATATTGT TAAGACCACT CATGAAGGCT ACTGCATTAG GGTTTTGAGT 180AAAAGAATCC CTTCCAAGTA AGTATGGGCT GCCGGTACGA GCCAAGGAGT TGCTGGTTTT 240TTCGGAAAGA CCATGACCGT GGATAACAAA CTCGTATTCC CAACGAAGGA TTTTACCAGT 300TTGCAACTGT GGGAGGCGTA GCTTTTGAGC AAAAACGAAG CATATAATAG CTAAACACAT 360ACCGCCGACC AAATCTACAA AGTAGTGGTG GGTAAGGTAC ATAGTACACC AGCAAAGCCA 420TAGAACATAT CCATAAAAGC AGAAGCGGTA TCGAGGAAAC ACATGCGAAA GGAAAAGTGC 480TTCCAGCATG GCCCATCCAG CGTGAAGAGA TGGAAAGGCA CCAAAAACAA CCGGAGAGTT 540AGAAAAACCA TCAGTGTAAA TGCTAGTGCC GAAGAGAGCA TCAATACGGG CCAATCCACC 600AGGAGAGCCA CGTACTGCAT ACGTGGCAGG TTCTAAACCA TACATATTTT CATACCAAGG 660AGGAGAACAG GGGAAAGCCA TTTGGATAAG AACACCAAAT AAATTCATAT AACCAAAAGT 720TCGAGCCCAA ACTGGAAGAG TTCCAGGAGG TGCAAAGATG AAAAGAATAA ATGAAATGAT 780AAAAGGAGCC GAATAATGCA TGACTCCATA TGGAACCCAG GCCAAAATAT CAAGGATGCT 840ATGCGTGGTT TTCGAGAGAA GACTAGAAAG ATTAGAGCCA TAAAGAATAT TTTCAAGTGT 900GGGTAAAACA CGAACCCATA TGGGTGGACG CCAGCGTTCT GGAATAAACC TACAAGAGTA 960AAATAAAATT GCCCAGGTGA TGATAACAAT GGCAGGAAAA AAAATTTGGC GTGTTAAAGG 1020AACGGTCAAC GCAATGGCCA AAAGACAGGC AATGCCAAAT TTCCCCCAGA ATCCAGGAGA 1080TTCAATGACA ATACAAGCAA AAATCAAATT ACCTGCTAGA AACACATATT GCAAATGTGT 1140CCATGACCAT TTCGTATTGC GTAGCAAACG AAATGTAGGC ATAGGGTTTA AGCTTGTTTC 1200CAACTTGTAT TGGGATGCTC GGTTACACGC AGCAAGGCGC TTTTTTAAGG TCGAAAGAGC 1260AGACATTGCT TCAAAGAATT ATCAGAGTAA AAAAGGGAAG CGTACGAAAA AAATTTCGTA 1320AGGAATTAAC CGGAAAACTA AAGGAAAAAA AAGGAATTTT TATGAAGGAA AGAAAGTAGC 1380TATTAAATGC AAGTGTCAAG CACTTAAAAG TAGCGATGTA AAATATTTAA AAAAAGATGG 1440ACCGATTAAC CAATGTTCAG CTCACAGTTG CCAGCAATCA GGGCTATTTT TTTATTTTTT 1500TTATAAAATT GCTAATTATA TATAATATAA TTAGTTTATT AACTTGCTTT TCCTCAAAAA 1560ACCAATTCGA GAAAGGAACT TTTGCAGAGG CAAAAAAGCT T 1601 1601 bases nucleicacid double linear mRNA Yes unknown 32 AGGAAGAUGA CUUGCAUCAA AGAUGGAGGAAGUGGUACUG GCAGGACGAU CAAUCAAAUC 60 AGCAGCAGGA CUAGGUAACG GCUCAGGUGAUGAUGAACCC ACGGACCAUU CAUGAUCGGU 120 GUUAGCAAGU UCCAUAUUGU UAAGACCACUCAUGAAGGCU ACUGCAUUAG GGUUUUGAGU 180 AAAAGAAUCC CUUCCAAGUA AGUAUGGGCUGCCGGUACGA GCCAAGGAGU UGCUGGUUUU 240 UUCGGAAAGA CCAUGACCGU GGAUAACAAACUCGUAUUCC CAACGAAGGA UUUUACCAGU 300 UUGCAACUGU GGGAGGCGUA GCUUUUGAGCAAAAACGAAG CAUAUAAUAG CUAAACACAU 360 ACCGCCGACC AAAUCUACAA AGUAGUGGUGGGUAAGGUAC AUAGUACACC AGCAAAGCCA 420 UAGAACAUAU CCAUAAAAGC AGAAGCGGUAUCGAGGAAAC ACAUGCGAAA GGAAAAGUGC 480 UUCCAGCAUG GCCCAUCCAG CGUGAAGAGAUGGAAAGGCA CCAAAAACAA CCGGAGAGUU 540 AGAAAAACCA UCAGUGUAAA UGCUAGUGCCGAAGAGAGCA UCAAUACGGG CCAAUCCACC 600 AGGAGAGCCA CGUACUGCAU ACGUGGCAGGUUCUAAACCA UACAUAUUUU CAUACCAAGG 660 AGGAGAACAG GGGAAAGCCA UUUGGAUAAGAACACCAAAU AAAUUCAUAU AACCAAAAGU 720 UCGAGCCCAA ACUGGAAGAG UUCCAGGAGGUGCAAAGAUG AAAAGAAUAA AUGAAAUGAU 780 AAAAGGAGCC GAAUAAUGCA UGACUCCAUAUGGAACCCAG GCCAAAAUAU CAAGGAUGCU 840 AUGCGUGGUU UUCGAGAGAA GACUAGAAAGAUUAGAGCCA UAAAGAAUAU UUUCAAGUGU 900 GGGUAAAACA CGAACCCAUA UGGGUGGACGCCAGCGUUCU GGAAUAAACC UACAAGAGUA 960 AAAUAAAAUU GCCCAGGUGA UGAUAACAAUGGCAGGAAAA AAAAUUUGGC GUGUUAAAGG 1020 AACGGUCAAC GCAAUGGCCA AAAGACAGGCAAUGCCAAAU UUCCCCCAGA AUCCAGGAGA 1080 UUCAAUGACA AUACAAGCAA AAAUCAAAUUACCUGCUAGA AACACAUAUU GCAAAUGUGU 1140 CCAUGACCAU UUCGUAUUGC GUAGCAAACGAAAUGUAGGC AUAGGGUUUA AGCUUGUUUC 1200 CAACUUGUAU UGGGAUGCUC GGUUACACGCAGCAAGGCGC UUUUUUAAGG UCGAAAGAGC 1260 AGACAUUGCU UCAAAGAAUU AUCAGAGUAAAAAAGGGAAG CGUACGAAAA AAAUUUCGUA 1320 AGGAAUUAAC CGGAAAACUA AAGGAAAAAAAAGGAAUUUU UAUGAAGGAA AGAAAGUAGC 1380 UAUUAAAUGC AAGUGUCAAG CACUUAAAAGUAGCGAUGUA AAAUAUUUAA AAAAAGAUGG 1440 ACCGAUUAAC CAAUGUUCAG CUCACAGUUGCCAGCAAUCA GGGCUAUUUU UUUAUUUUUU 1500 UUAUAAAAUU GCUAAUUAUA UAUAAUAUAAUUAGUUUAUU AACUUGCUUU UCCUCAAAAA 1560 ACCAAUUCGA GAAAGGAACU UUUGCAGAGGCAAAAAAGCU U 1601 12 amino acids amino acid single linear peptideunknown 33 Cys Phe Thr Ser Ser Tyr Phe Pro Asp Asp Arg Arg 5 10 19 aminoacids amino acid single linear peptide unknown 34 Cys Tyr Thr Ser IleGlu Lys Tyr Asp Ile Ser Lys Ser Asp Pro 5 10 15 Leu Ala Ala Asp 1553bases nucleic acid double linear Genomic DNA unknown 35 TTTTACATATATTATTCACC CAATATCATA ACAAAAACAA ACTGAATGAT GGCATCTTCT 60 ATTTTGCGTTCCAAAATAAT ACAAAAACCG TACCAATTAT TCCACTACTA TTTTCTTCTG 120 GAGAAGGCTCCTGGTTCTAC AGTTAGTGAT TTGAATTTTG ATACAAACAT ACAAACGAGT 180 TTACGTAAATTAAAGCATCA TCATTGGACG GTGGGAGAAA TATTCCATTA TGGGTTTTTG 240 GTTTCCATACTTTTTTTCGT GTTTGTGGTT TTCCCAGCTT CATTTTTTAT AAAATTACCA 300 ATAATCTTAGCATTTGCTAC TTGTTTTTTA ATACCCTTAA CATCACAATT TTTTCTTCCT 360 GCCTTGCCCGTTTTCACTTG GTTGGCATTA TATTTTACGT GTGCTAAAAT ACCTCAAGAA 420 TGGAAACCAGCTATCACAGT TAAAGTTTTA CCAGCTATGG AAACAATTTT GTACGGCGAT 480 AATTTATCAAATGTTTTGGC AACCATCACT ACCGGAGTGT TAGATATATT GGCATGGTTA 540 CCATATGGGATTATTCATTT CAGTTTCCCA TTTGTACTTG CTGCTATTAT ATTTTTATTT 600 GGGCCACCGACGGCATTAAG ATCATTTGGA TTTGCCTTTG GTTATATGAA CTTGCTTGGA 660 GTCTTGATTCAAATGGCATT CCCAGCTGCT CCTCCATGGT ACAAAAACTT GCACGGATTA 720 GAACCAGCTAATTATTCAAT GCACGGGTCT CCTGGTGGAC TTGGAAGGAT AGATAAATTG 780 TTAGGTGTTGATATGTATAC CACAGGGTTT TCCAATTCAT CAATCATTTT TGGGGCATTC 840 CCATCGTTACATTCAGGATG TTGTATCATG GAAGTGTTAT TTTTGTGTTG GTTGTTTCCA 900 CGATTCAAGTTTGTGTGGGT TACATACGCA TCTTGGCTTT GGTGGAGCAC GATGTATTTG 960 ACCCATCACTACTTTGTCGA TTTGATTGGT GGAGCCATGC TATCTTTGAC TGTTTTTGAA 1020 TTCACCAAATATAAATATTT GCCAAAAAAC AAAGAAGGCC TTTTCTGTCG TTGGTCATAC 1080 ACTGAAATTGAAAAAATCGA TATCCAAGAG ATTGACCCTT TATCATACAA TTATATCCCT 1140 GTCAACAGCAATGATAATGA AAGCAGATTG TATACGAGAG TGTACCAAGA GCCTCAGGTT 1200 AGTCCCCCACAGAGAGCTGA AACACCTGAA GCATTTGAGA TGTCAAATTT TTCTAGGTCT 1260 AGACAAAGCTCAAAGACTCA GGTTCCATTG AGTAATCTTA CTAACAATGA TCAAGTGCCT 1320 GGAATTAACGAAGAGGATGA AGAAGAAGAA GGCGATGAAA TTTCGTCGAG TACTCCTTCG 1380 GTGTTTGAAGACGAACCACA GGGTAGCACA TATGCTGCAT CCTCAGCTAC ATCAGTAGAT 1440 GATTTGGATTCCAAAAGAAA TTAGTAAAAC AGCAGTTTCT ATTAATTTCT TTATTTCCTC 1500 CTAATTAATGATTTTATGTT CAATACCTAC ACTATCTGTT TTTAATTTCC TAC 1553 472 amino acidsamino acid single linear peptide unknown 36 Met Met Ala Ser Ser Ile LeuArg Ser Lys Ile Ile Gln Lys Pro 1 5 10 15 Tyr Gln Leu Phe His Tyr TyrPhe Leu Leu Glu Lys Ala Pro Gly 20 25 30 Ser Thr Val Ser Asp Leu Asn PheAsp Thr Asn Ile Gln Thr Ser 35 40 45 Leu Arg Lys Leu Lys His His His TrpThr Val Gly Glu Ile Phe 50 55 60 His Tyr Gly Phe Leu Val Ser Ile Leu PhePhe Val Phe Val Val 65 70 75 Phe Pro Ala Ser Phe Phe Ile Lys Leu Pro IleIle Leu Ala Phe 80 85 90 Ala Thr Cys Phe Leu Ile Pro Leu Thr Ser Gln PhePhe Leu Pro 95 100 105 Ala Leu Pro Val Phe Thr Trp Leu Ala Leu Tyr PheThr Cys Ala 110 115 120 Lys Ile Pro Gln Glu Trp Lys Pro Ala Ile Thr ValLys Val Leu 125 130 135 Pro Ala Met Glu Thr Ile Leu Tyr Gly Asp Asn LeuSer Asn Val 140 145 150 Leu Ala Thr Ile Thr Thr Gly Val Leu Asp Ile LeuAla Trp Leu 155 160 165 Pro Tyr Gly Ile Ile His Phe Ser Phe Pro Phe ValLeu Ala Ala 170 175 180 Ile Ile Phe Leu Phe Gly Pro Pro Thr Ala Leu ArgSer Phe Gly 185 190 195 Phe Ala Phe Gly Tyr Met Asn Leu Leu Gly Val LeuIle Gln Met 200 205 210 Ala Phe Pro Ala Ala Pro Pro Trp Tyr Lys Asn LeuHis Gly Leu 215 220 225 Glu Pro Ala Asn Tyr Ser Met His Gly Ser Pro GlyGly Leu Gly 230 235 240 Arg Ile Asp Lys Leu Leu Gly Val Asp Met Tyr ThrThr Gly Phe 245 250 255 Ser Asn Ser Ser Ile Ile Phe Gly Ala Phe Pro SerLeu His Ser 260 265 270 Gly Cys Cys Ile Met Glu Val Leu Phe Leu Cys TrpLeu Phe Pro 275 280 285 Arg Phe Lys Phe Val Trp Val Thr Tyr Ala Ser TrpLeu Trp Trp 290 295 300 Ser Thr Met Tyr Leu Thr His His Tyr Phe Val AspLeu Ile Gly 305 310 315 Gly Ala Met Leu Ser Leu Thr Val Phe Glu Phe ThrLys Tyr Lys 320 325 330 Tyr Leu Pro Lys Asn Lys Glu Gly Leu Phe Cys ArgTrp Ser Tyr 335 340 345 Thr Glu Ile Glu Lys Ile Asp Ile Gln Glu Ile AspPro Leu Ser 350 355 360 Tyr Asn Tyr Ile Pro Val Asn Ser Asn Asp Asn GluSer Arg Leu 365 370 375 Tyr Thr Arg Val Tyr Gln Glu Pro Gln Val Ser ProPro Gln Arg 380 385 390 Ala Glu Thr Pro Glu Ala Phe Glu Met Ser Asn PheSer Arg Ser 395 400 405 Arg Gln Ser Ser Lys Thr Gln Val Pro Leu Ser AsnLeu Thr Asn 410 415 420 Asn Asp Gln Val Pro Gly Ile Asn Glu Glu Asp GluGlu Glu Glu 425 430 435 Gly Asp Glu Ile Ser Ser Ser Thr Pro Ser Val PheGlu Asp Glu 440 445 450 Pro Gln Gly Ser Thr Tyr Ala Ala Ser Ser Ala ThrSer Val Asp 455 460 465 Asp Leu Asp Ser Lys Arg Asn 470 22 bases nucleicacid single linear Other nucleic acid (synthetic DNA) unknown 37GACTATTTCA TTATGGGGCC CC 22 30 bases nucleic acid single linear Othernucleic acid (synthetic DNA) unknown 38 GTTAACTCGA GAAAGTGCCC ATCAGTGTTC30 29 bases nucleic acid single linear Other nucleic acid (syntheticDNA) unknown 39 GTTAACGGTA CCTCATCGTT ACACCGTTC 29 19 bases nucleic acidsingle linear Other nucleic acid (synthetic DNA) unknown 40 GCTAAACGACAATCCTGAC 19 21 bases nucleic acid single linear Other nucleic acid(synthetic DNA) unknown 41 CGTTGGCCGA TTCATTAATG C 21 401 amino acidsamino acid single linear peptide unknown 42 Met Ala Asn Pro Phe Ser ArgTrp Phe Leu Ser Glu Arg Pro Pro 1 5 10 15 Asn Cys His Val Ala Asp LeuGlu Thr Ser Leu Asp Pro His Gln 20 25 30 Thr Leu Leu Lys Val Gln Lys TyrLys Pro Ala Leu Ser Asp Trp 35 40 45 Val His Tyr Ile Phe Leu Gly Ser IleMet Leu Phe Val Phe Ile 50 55 60 Thr Asn Pro Ala Pro Trp Ile Phe Lys IleLeu Phe Tyr Cys Phe 65 70 75 Leu Gly Thr Leu Phe Ile Ile Pro Ala Thr SerGln Phe Phe Phe 80 85 90 Asn Ala Leu Pro Ile Leu Thr Trp Val Ala Leu TyrPhe Thr Ser 95 100 105 Ser Tyr Phe Pro Asp Asp Arg Arg Pro Pro Ile ThrVal Lys Val 110 115 120 Leu Pro Ala Val Glu Thr Ile Leu Tyr Gly Asp AsnLeu Ser Asp 125 130 135 Ile Leu Ala Thr Ser Thr Asn Ser Phe Leu Asp IleLeu Ala Trp 140 145 150 Leu Pro Tyr Gly Leu Phe His Phe Gly Ala Pro PheVal Val Ala 155 160 165 Ala Ile Leu Phe Val Phe Gly Pro Pro Thr Val LeuGln Gly Tyr 170 175 180 Ala Phe Ala Phe Gly Tyr Met Asn Leu Phe Gly ValIle Met Gln 185 190 195 Asn Val Phe Pro Ala Ala Pro Pro Trp Tyr Lys IleLeu Tyr Gly 200 205 210 Leu Gln Ser Ala Asn Tyr Asp Met His Gly Ser ProGly Gly Leu 215 220 225 Ala Arg Ile Asp Lys Leu Leu Gly Ile Asn Met TyrThr Thr Cys 230 235 240 Phe Ser Asn Ser Ser Val Ile Phe Gly Ala Phe ProSer Leu His 245 250 255 Ser Gly Cys Ala Thr Met Glu Ala Leu Phe Phe CysTyr Cys Phe 260 265 270 Pro Lys Leu Lys Pro Leu Phe Ile Ala Tyr Val CysTrp Leu Trp 275 280 285 Trp Ser Thr Met Tyr Leu Thr His His Tyr Phe ValAsp Leu Met 290 295 300 Ala Gly Ser Val Leu Ser Tyr Val Ile Phe Gln TyrThr Lys Tyr 305 310 315 Thr His Leu Pro Ile Val Asp Thr Ser Leu Phe CysArg Trp Ser 320 325 330 Tyr Thr Ser Ile Glu Lys Tyr Asp Ile Ser Lys SerAsp Pro Leu 335 340 345 Ala Ala Asp Ser Asn Asp Ile Glu Ser Val Pro LeuSer Asn Leu 350 355 360 Glu Leu Asp Phe Asp Leu Asn Met Thr Asp Glu ProSer Val Ser 365 370 375 Pro Ser Leu Phe Asp Gly Ser Thr Ser Val Ser ArgSer Ser Ala 380 385 390 Thr Ser Ile Thr Ser Leu Gly Val Lys Arg Ala 395400 401 amino acids amino acid single linear peptide unknown 43 Met AlaAsn Pro Phe Ser Arg Trp Phe Leu Ser Glu Arg Pro Pro 1 5 10 15 Asn CysHis Val Ala Asp Leu Glu Thr Ser Leu Asp Pro His Gln 20 25 30 Thr Leu LeuLys Val Gln Lys Tyr Lys Pro Ala Leu Ser Asp Trp 35 40 45 Val His Tyr IlePhe Leu Gly Ser Ile Met Leu Phe Val Phe Ile 50 55 60 Thr Asn Pro Ala ProTrp Ile Phe Lys Ile Leu Phe Tyr Cys Phe 65 70 75 Leu Gly Thr Leu Phe IleIle Pro Ala Thr Ser Gln Phe Phe Phe 80 85 90 Asn Ala Leu Pro Ile Leu ThrTrp Val Ala Leu Tyr Phe Thr Ser 95 100 105 Ser Tyr Phe Pro Asp Asp ArgArg Pro Pro Ile Thr Val Lys Val 110 115 120 Leu Pro Ala Val Glu Thr IleLeu Tyr Gly Asp Asn Leu Ser Asp 125 130 135 Ile Leu Ala Thr Ser Thr AsnSer Phe Leu Asp Ile Leu Ala Trp 140 145 150 Leu Pro Tyr Gly Leu Phe HisTyr Gly Ala Pro Phe Val Val Ala 155 160 165 Ala Ile Leu Phe Val Phe GlyPro Pro Thr Val Leu Gln Gly Tyr 170 175 180 Ala Phe Ala Phe Gly Tyr MetAsn Leu Phe Gly Val Ile Met Gln 185 190 195 Asn Val Phe Pro Ala Ala ProPro Trp Tyr Lys Ile Leu Tyr Gly 200 205 210 Leu Gln Ser Ala Asn Tyr AspMet His Gly Ser Pro Gly Gly Leu 215 220 225 Ala Arg Ile Asp Lys Leu LeuGly Ile Asn Met Tyr Thr Thr Cys 230 235 240 Phe Ser Asn Ser Ser Val IlePhe Gly Ala Phe Pro Ser Leu His 245 250 255 Ser Gly Cys Ala Thr Met GluAla Leu Phe Phe Cys Tyr Cys Phe 260 265 270 Pro Lys Leu Lys Pro Leu PheIle Ala Tyr Val Cys Trp Leu Trp 275 280 285 Trp Ser Thr Met Tyr Leu ThrHis His Tyr Phe Val Asp Leu Met 290 295 300 Ala Gly Ser Val Leu Ser TyrVal Ile Phe Gln Tyr Thr Lys Tyr 305 310 315 Thr His Leu Pro Ile Val AspThr Ser Leu Phe Cys Arg Trp Ser 320 325 330 Tyr Thr Ser Ile Glu Lys TyrAsp Ile Ser Lys Ser Asp Pro Leu 335 340 345 Ala Ala Asp Ser Asn Asp IleGlu Ser Val Pro Leu Ser Asn Leu 350 355 360 Glu Leu Asp Phe Asp Leu AsnMet Thr Asp Glu Pro Ser Val Ser 365 370 375 Pro Ser Leu Phe Asp Gly SerThr Ser Val Ser Arg Ser Ser Ala 380 385 390 Thr Ser Ile Thr Ser Leu GlyVal Lys Arg Ala 395 400 1206 base pairs nucleic acid double linearGenomic DNA unknown 44 ATGGCAAACC CTTTTTCGAG ATGGTTTCTA TCAGAGAGACCTCCAAACTG CCATGTAGCC 60 GATTTAGAAA CAAGTTTAGA TCCCCATCAA ACGTTGTTGAAGGTGCAAAA ATACAAACCC 120 GCTTTAAGCG ACTGGGTGCA TTACATCTTC TTGGGATCCATCATGCTGTT TGTGTTCATT 180 ACTAATCCCG CACCTTGGAT CTTCAAGATC CTTTTTTATTGTTTCTTGGG CACTTTATTC 240 ATCATTCCAG CTACGTCACA GTTTTTCTTC AATGCCTTGCCCATCCTAAC ATGGGTGGCG 300 CTGTATTTCA CTTCATCGTA CTTTCCAGAT GACCGCAGGCCTCCTATTAC TGTCAAAGTG 360 TTACCAGCGG TGGAAACAAT TTTATACGGC GACAATTTAAGTGATATTCT TGCAACATCG 420 ACGAATTCCT TTTTGGACAT TTTAGCATGG TTACCGTACGGACTATTTCA TTTTGGGGCC 480 CCATTTGTCG TTGCTGCCAT CTTATTCGTA TTTGGTCCACCAACTGTTTT GCAAGGTTAT 540 GCTTTTGCAT TTGGTTATAT GAACCTGTTT GGTGTTATCATGCAAAATGT CTTTCCAGCC 600 GCTCCCCCAT GGTATAAAAT TCTCTATGGA TTGCAATCAGCCAACTATGA TATGCATGGC 660 TCGCCTGGTG GATTAGCTAG AATTGATAAG CTACTCGGTATTAATATGTA TACTACATGT 720 TTTTCAAATT CCTCCGTCAT TTTCGGTGCT TTTCCTTCACTGCATTCCGG GTGTGCTACT 780 ATGGAAGCCC TGTTTTTCTG TTATTGTTTT CCAAAATTGAAGCCCTTGTT TATTGCTTAT 840 GTTTGCTGGT TATGGTGGTC AACTATGTAT CTGACACACCATTATTTTGT AGACCTTATG 900 GCAGGTTCTG TGCTGTCATA CGTTATTTTC CAGTACACAAAGTACACACA TTTACCAATT 960 GTAGATACAT CTCTTTTTTG CAGATGGTCA TACACTTCAATTGAGAAATA CGATATATCA 1020 AAGAGTGATC CATTGGCTGC AGATTCAAAC GATATCGAAAGTGTCCCTTT GTCCAACTTG 1080 GAACTTGACT TTGATCTTAA TATGACTGAT GAACCCAGTGTAAGCCCTTC GTTATTTGAT 1140 GGATCTACTT CTGTTTCTCG TTCGTCCGCC ACGTCTATAACGTCACTAGG TGTAAAGAGG 1200 GCTTAA 1206 1206 base pairs nucleic aciddouble linear Genomic DNA unknown 45 ATGGCAAACC CTTTTTCGAG ATGGTTTCTATCAGAGAGAC CTCCAAACTG CCATGTAGCC 60 GATTTAGAAA CAAGTTTAGA TCCCCATCAAACGTTGTTGA AGGTGCAAAA ATACAAACCC 120 GCTTTAAGCG ACTGGGTGCA TTACATCTTCTTGGGATCCA TCATGCTGTT TGTGTTCATT 180 ACTAATCCCG CACCTTGGAT CTTCAAGATCCTTTTTTATT GTTTCTTGGG CACTTTATTC 240 ATCATTCCAG CTACGTCACA GTTTTTCTTCAATGCCTTGC CCATCCTAAC ATGGGTGGCG 300 CTGTATTTCA CTTCATCGTA CTTTCCAGATGACCGCAGGC CTCCTATTAC TGTCAAAGTG 360 TTACCAGCGG TGGAAACAAT TTTATACGGCGACAATTTAA GTGATATTCT TGCAACATCG 420 ACGAATTCCT TTTTGGACAT TTTAGCATGGTTACCGTACG GACTATTTCA TTATGGGGCC 480 CCATTTGTCG TTGCTGCCAT CTTATTCGTATTTGGTCCAC CAACTGTTTT GCAAGGTTAT 540 GCTTTTGCAT TTGGTTATAT GAACCTGTTTGGTGTTATCA TGCAAAATGT CTTTCCAGCC 600 GCTCCCCCAT GGTATAAAAT TCTCTATGGATTGCAATCAG CCAACTATGA TATGCATGGC 660 TCGCCTGGTG GATTAGCTAG AATTGATAAGCTACTCGGTA TTAATATGTA TACTACATGT 720 TTTTCAAATT CCTCCGTCAT TTTCGGTGCTTTTCCTTCAC TGCATTCCGG GTGTGCTACT 780 ATGGAAGCCC TGTTTTTCTG TTATTGTTTTCCAAAATTGA AGCCCTTGTT TATTGCTTAT 840 GTTTGCTGGT TATGGTGGTC AACTATGTATCTGACACACC ATTATTTTGT AGACCTTATG 900 GCAGGTTCTG TGCTGTCATA CGTTATTTTCCAGTACACAA AGTACACACA TTTACCAATT 960 GTAGATACAT CTCTTTTTTG CAGATGGTCATACACTTCAA TTGAGAAATA CGATATATCA 1020 AAGAGTGATC CATTGGCTGC AGATTCAAACGATATCGAAA GTGTCCCTTT GTCCAACTTG 1080 GAACTTGACT TTGATCTTAA TATGACTGATGAACCCAGTG TAAGCCCTTC GTTATTTGAT 1140 GGATCTACTT CTGTTTCTCG TTCGTCCGCCACGTCTATAA CGTCACTAGG TGTAAAGAGG 1200 GCTTAA 1206 1206 base pairsnucleic acid double linear Genomic DNA unknown 46 ATGGCAAACC CTTTTTCGAGATGGTTTCTA TCAGAGAGAC CTCCAAACTG CCATGTAGCC 60 GATTTAGAAA CAAGTTTAGATCCCCATCAA ACGTTGTTGA AGGTGCAAAA ATACAAACCC 120 GCTTTAAGCG ACTGGGTGCATTACATCTTC TTGGGATCCA TCATGCTGTT TGTGTTCATT 180 ACTAATCCCG CACCTTGGATCTTCAAGATC CTTTTTTATT GTTTCTTGGG CACTTTATTC 240 ATCATTCCAG CTACGTCACAGTTTTTCTTC AATGCCTTGC CCATCCTAAC ATGGGTGGCG 300 CTGTATTTCA CTTCATCGTACTTTCCAGAT GACCGCAGGC CTCCTATTAC TGTCAAAGTG 360 TTACCAGCGG TGGAAACAATTTTATACGGC GACAATTTAA GTGATATTCT TGCAACATCG 420 ACGAATTCCT TTTTGGACATTTTAGCATGG TTACCGTACG GACTATTTCA TTATGGGGCC 480 CCATTTGTCG TTGCTGCCATCTTATTCGTA TTTGGTCCAC CAACTGTTTT GCAAGGTTAT 540 GCTTTTGCAT TTGGTTATATGAACCTGTTT GGTGTTATCA TGCAAAATGT CTTTCCAGCC 600 GCTCCCCCAT GGTATAAAATTCTCTATGGA TTGCAATCAG CCAACTATGA TATGCATGGC 660 TCGCCTGGTG GATTAGCTAGAATTGATAAG CTACTCGGTA TTAATATGTA TACTACAGCT 720 TTTTCAAATT CCTCCGTCATTTTCGGTGCT TTTCCTTCAC TGCATTCCGG GTGTGCTACT 780 ATGGAAGCCC TGTTTTTCTGTTATTGTTTT CCAAAATTGA AGCCCTTGTT TATTGCTTAT 840 GTTTGCTGGT TATGGTGGTCAACTATGTAT CTGACACACC ATTATTTTGT AGACCTTATG 900 GCAGGTTCTG TGCTGTCATACGTTATTTTC CAGTACACAA AGTACACACA TTTACCAATT 960 GTAGATACAT CTCTTTTTTGCAGATGGTCA TACACTTCAA TTGAGAAATA CGATATATCA 1020 AAGAGTGATC CATTGGCTGCAGATTCAAAC GATATCGAAA GTGTCCCTTT GTCCAACTTG 1080 GAACTTGACT TTGATCTTAATATGACTGAT GAACCCAGTG TAAGCCCTTC GTTATTTGAT 1140 GGATCTACTT CTGTTTCTCGTTCGTCCGCC ACGTCTATAA CGTCACTAGG TGTAAAGAGG 1200 GCTTAA 1206 1206 basepairs nucleic acid double linear Genomic DNA unknown 47 ATGGCAAACCCTTTTTCGAG ATGGTTTCTA TCAGAGAGAC CTCCAAACTG CCATGTAGCC 60 GATTTAGAAACAAGTTTAGA TCCCCATCAA ACGTTGTTGA AGGTGCAAAA ATACAAACCC 120 GCTTTAAGCGACTGGGTGCA TTACATCTTC TTGGGATCCA TCATGCTGTT TGTGTTCATT 180 ACTAATCCCGCACCTTGGAT CTTCAAGATC CTTTTTTATT GTTTCTTGGG CACTTTATTC 240 ATCATTCCAGCTACGTCACA GTTTTTCTTC AATGCCTTGC CCATCCTAAC ATGGGTGGCG 300 CTGTATTTCACTTCATCGTA CTTTCCAGAT GACCGCAGGC CTCCTATTAC TGTCAAAGTG 360 TTACCAGCGGTGGAAACAAT TTTATACGGC GACAATTTAA GTGATATTCT TGCAACATCG 420 ACGAATTCCTTTTTGGACAT TTTAGCATGG TTACCGTACG GACTATTTCA TTTTGGGGCC 480 CCATTTGTCGTTGCTGCCAT CTTATTCGTA TTTGGTCCAC CAACTGTTTT GCAAGGTTAT 540 GCTTTTGCATTTGGTTATAT GAACCTGTTT GGTGTTATCA TGCAAAATGT CTTTCCAGCC 600 GCTCCCCCATGGTATAAAAT TCTCTATGGA TTGCAATCAG CCAACTATGA TATGCATGGC 660 TCGCCTGGTGGATTAGCTAG AATTGATAAG CTACTCGGTA TTAATATGTA TACTACAGCT 720 TTTTCAAATTCCTCCGTCAT TTTCGGTGCT TTTCCTTCAC TGCATTCCGG GTGTGCTACT 780 ATGGAAGCCCTGTTTTTCTG TTATTGTTTT CCAAAATTGA AGCCCTTGTT TATTGCTTAT 840 GTTTGCTGGTTATGGTGGTC AACTATGTAT CTGACACACC ATTATTTTGT AGACCTTATG 900 GCAGGTTCTGTGCTGTCATA CGTTATTTTC CAGTACACAA AGTACACACA TTTACCAATT 960 GTAGATACATCTCTTTTTTG CAGATGGTCA TACACTTCAA TTGAGAAATA CGATATATCA 1020 AAGAGTGATCCATTGGCTGC AGATTCAAAC GATATCGAAA GTGTCCCTTT GTCCAACTTG 1080 GAACTTGACTTTGATCTTAA TATGACTGAT GAACCCAGTG TAAGCCCTTC GTTATTTGAT 1140 GGATCTACTTCTGTTTCTCG TTCGTCCGCC ACGTCTATAA CGTCACTAGG TGTAAAGAGG 1200 GCTTAA 120630 bases nucleic acid single linear Other nucleic acid (synthetic DNA)unknown 48 AATATGTATA CTACATGTTT TTCAAATTCC 30 30 bases nucleic acidsingle linear Other nucleic acid (synthetic DNA) unknown 49 GTTAACTCGAGAAAGTGCCC ATCAGTGTTC 30 29 bases nucleic acid single linear Othernucleic acid (synthetic DNA) unknown 50 GTTAACGGTA CCAGAGGAAA GAATAACGC29

What is claimed is:
 1. An isolated DNA comprising a nucleic acidsequence which encodes the amino acid sequence of SEQ ID No. 2, 4, or 5.2. An isolated DNA comprising the nucleic acid sequence of SEQ ID No. 1,3, or
 12. 3. The DNA according to claim 1, which is isolated from mold.4. The DNA according to claim 2, which is isolated from mold.
 5. The DNAaccording to claim 1, which is isolated from an microorganism belongingto the genus Aspergillus.
 6. The DNA according to claim 2, which isisolated from a microorganism belonging to the genus Aspergillus.
 7. Anisolated DNA comprising a nucleic acid sequence which encodes a portionof the amino acid sequence of SEQ ID No. 2 and which portion confersupon a cell resistance to aureobasidin.
 8. An isolated DNA comprising afragment of the nucleic acid sequence of SEQ ID No. 1 and which fragmentencodes a protein which confers upon a cell resistance to aureobasidin.9. An isolated DNA comprising a nucleic acid sequence which encodes theamino acid sequence of SEQ ID No. 2 or containing one conservativesubstitution thereof and which amino acid sequence confers upon a cellresistance to aureobasidin.
 10. An isolated DNA comprising the nucleicacid sequence of SEQ ID No. 1 or containing one mutation thereof andwhich encodes a protein which confers upon a cell resistance toaureobasidin.
 11. An isolated DNA which is hybridizable with the DNAaccording to claim 1, 2, 7, 8, 9, or 10 or its full complement underconditions of 6×SSC, 1% of sodium lauryl sulfate, 100 μg/ml of salmonsperm DNA and 5×Denhardt's solution at 65° C. for 20 hours and whichencodes a protein which confers upon a cell sensitivity or resistance toaureobasidin.
 12. An isolated DNA, which comprises a DNA having asequence which is fully complementary to the DNA according to claim 1,2, 7, 8, 9, 10, or
 11. 13. The isolated DNA according to claim 12, whichhas the nucleic acid sequence according to SEQ ID No.
 13. 14. Anisolated RNA, which comprises an RNA having a sequence which is fullycomplementary to the mRNA encoded by the DNA according to claim 1, 2, 7,8, 9, 10, or
 11. 15. The isolated RNA according to claim 14, which hasthe nucleic acid sequence according to SEQ ID No. 14, or a fragment of15 or more bases thereof.
 16. A recombinant plasmid comprising a nucleicacid sequence containing the DNA according to claim 1, 2, 7, 8, 9, 10,or
 11. 17. A transformant containing the recombinant plasmid accordingto claim
 16. 18. A process for producing a protein, which comprises:incubating the transformant according to claim 17 under conditions toexpress the protein, and recovering the protein from the culture medium.19. An isolated DNA comprising a nucleic acid sequence which encodes aprotein which has an amino acid sequence according to SEQ ID No. 4except that the 275th amino acid residue Gly in the amino acid sequenceis replaced by another amino acid residue, and which confers upon a cellresistance to aureobasidin.
 20. A chromosome integration vector for ahost fungus or mold, which comprises a nucleic acid sequence containingthe DNA according to claim 1, 2, 7, 8, 9, 10, 11, or
 19. 21. A processfor producing an aureobasidin resistant transformant, which comprises:(a) providing a replication vector which contains a DNA according toclaim 1, 2, 7, 8, 9, 10, 11 or 19 and which encodes a protein impartingaureobasidin resistance, (b) cleaving the DNA in the replication vectorat one site to produce a chromosome integration vector for a host fungusor mold; (c) integrating the chromosome integration vector into thechromosome of the host; and (d) selecting a host which has beentransformed into an aureobasidin resistant transformant in the presenceof aureobasidin.
 22. A transformant obtained by the process according toclaim
 21. 23. An isolated DNA comprising a nucleic acid sequence whichencodes a portion of the amino acid sequence of SEQ ID No. 4 or 5 andwhich portion confers upon a cell sensitivity to aureobasidin.
 24. Anisolated DNA comprising a fragment of the nucleic acid sequence of SEQID No. 3 or 12 and which fragment encodes a protein which confers upon acell sensitivity to aureobasidin.
 25. An isolated DNA comprising anucleic acid sequence which encodes the amino acid sequence of SEQ IDNo. 4 or 5 or containing one conservative substitution thereof and whichamino acid sequence confers upon a cell sensitivity to aureobasidin. 26.An isolated DNA comprising the nucleic acid sequence of SEQ ID No. 3 or12 or containing one mutation thereof and which encodes a protein whichconfers upon a cell sensitivity to aureobasidin.
 27. An isolated DNAwhich is hybridizable with the DNA according to claim 23, 24, 25, or 26or its full complement under conditions of 6×SSC, 1% of sodium laurylsulfate, 100 μg/ml of salmon sperm DNA and 5×Denhardt's solution at 65°C. for 20 hours and which encodes a protein which confers upon a cellsensitivity or resistance to aureobasidin.
 28. An isolated DNA, whichcomprises a DNA having a sequence which is fully complementary to theDNA according to claim 23, 24, 25, 26 or
 27. 29. An isolated RNA, whichcomprises an RNA having a sequence which is fully complementary to themRNA encoded by the DNA according to claim 23, 24, 25, 26, or
 27. 30. Arecombinant plasmid comprising a nucleic acid sequence containing theDNA according to claim 23, 24, 25, 26, or
 27. 31. A transformantcontaining the recombinant plasmid according to claim
 30. 32. A processfor producing a protein, which comprises: incubating the transformantaccording to claim 31 under conditions to express the protein, andrecovering the protein from the culture medium.